Diagnosing genetic disorders

ABSTRACT

This invention relates to agents and conjugates that can be used to detect and isolate target components from complex mixtures such as nucleic acids from biological samples, cells from bodily fluids, and nascent proteins from translation reactions. Agents comprise a detectable moiety bound to a photoreactive moiety. Conjugates comprise agents coupled to substrates by covalent bounds which can be selectively cleaved with the administration of electromagnetic radiation. Targets substances labeled with detectable molecules can be easily identified and separated from a heterologous mixture of substances. Exposure of the conjugate to radiation releases the target in a functional form and completely unaltered. Using photocleavable molecular precursors as the conjugates, label can be incorporated into macromolecules, the nascent macromolecules isolated and the label completely removed. The invention also relates to targets isolated with these conjugates which may be useful as pharmaceutical agents or compositions that can be administered to humans and other mammals. Useful compositions include biological agents such as nucleic acids, proteins, lipids and cytokines. Conjugates can also be used to monitor the pathway and half-life of pharmaceutical composition in vivo and for diagnostic, therapeutic and prophylactic purposes. The invention also relates to kits comprised of agents and conjugates that can be used for the detection of diseases, disorders and nearly any individual substance in a complex background of substances.

REFERENCE TO RELATED APPLICATIONS

[0001] This patent application is a continuation-in-part of U.S. patentapplication Ser. No. 08/240,511, filed May 11, 1994.

RIGHTS IN THE INVENTION

[0002] This invention was made with United States Government supportunder grant number EM4727-03, awarded by the National Institutes ofHealth, and grant number DAAL03-92-G-0172, awarded by the Army ResearchOffice, and the United States Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] This invention relates to agents and conjugates used in thedetection and isolation of targets from heterologous mixtures. Agentscomprise a detectable moiety bound to a photoreactive moiety. Conjugatescomprise agents which are coupled to substrates by one or more covalentbonds. These bonds can be easily and selectively cleaved or photocleavedwith the application of electromagnetic radiation. Substrates which maybe coupled to agents include amino acids, peptides, proteins,nucleotides, nucleic acid primers for PCR reactions and lipids. Theinvention also relates to rapid and efficient methods for the detectionand isolation of targets, such as cells, nucleic acids and proteins, andto kits which contain these components.

[0005] 2. Description of the Background

[0006] Basic scientific techniques including some of the majorbreakthroughs in molecular biology, chemistry and medicine have certainfeatures in common. Two of these features are the specific detection andisolation of individual components from complex mixtures. For example,electrophoresis and chromatography are each widely utilized proceduresto detect or isolate macromolecules from biological samples. Theseprocedures take advantage of unique or identifiable molecular propertiesof the components to be isolated such as charge, hydrophobicity andmolecular weight, to characterize and identify macromolecules. Dependingon their method of isolation, macromolecules isolated can often beutilized as products in downstream processes.

[0007] Some of the more useful detection and isolation procedures takeadvantage of physical properties of the element of interest, thesubstrate, or of molecules which can be easily attached to substrates.One of the most widely used of these properties is radioactivity andradioactive labeling with radionuclides. For the most part, substancesare not naturally radioactive and can be labeled with radioactive atoms,referred to as radionuclides, and detected using standard and well-knownradiographic procedures. Radioactive elements are detectable becausethey emit large amounts of energy in the form of alpha, beta or gammarays as they decay. Radioactivity is generally useful for labelingbecause the label is not affected by the physical state or chemicalcombination of the substance to be labeled. In addition, the specificradiation emitted can be identified by the nature of the radiation (e.g.α,β or γ), its energy and the half-life of the process. Targets can beidentified in complex mixtures from the radiation profile emitted.Further, radioactively labeled substances can be followedradiographically in chemical pathways and biological systems.

[0008] Unfortunately, radioactivity is a hazard to both human health andthe environment. The protection which must be afforded each worker issubstantial. Special laboratory procedures, dedicated facilities andequipment, detailed record keeping and special training of laboratorypersonnel are all required for the safe use of radionuclides. Productionof radioactive reagents is also very expensive as is safe disposal whichdrives up the cost of all experiments involving radioactive agents.Further, under present guidelines, all users of radioactivity requirespecialized supervision and federal regulations must be strictly andcarefully adhered to requiring an enormous amount of record keeping.

[0009] Radioactive labeling methods also do not always provide a meansof isolating products in a form which can be further utilized. Thepresence of radioactivity compromises utility for further biochemical orbiophysical procedures in the laboratory and in animals. This is clearin the case of in vitro or in vivo expression of proteinsbiosynthetically labeled with radioactive amino acids or tagged withother radioactive markers. The harm or at least potential harm of theradioactivity outweighs the benefits which might be produced by theprotein composition.

[0010] Disposal of radioactive waste is also of increasing concern bothbecause of the potential risk to the public and the lack of radioactivewaste disposal sites. In addition, the use of radioactive labeling istime consuming, in some cases requiring as much as several days fordetection of the radioactive label. The long time needed for suchexperiments is a key consideration and can seriously impede researchproductivity. While faster methods of radioactive detection areavailable, they are expensive and often require complex imageenhancement devices.

[0011] There are many other detectable physical properties which canexist in chemicals and chemical moieties that can be used to detect andisolate substances. One of these physical properties is the property ofluminescence which includes the phenomena of fluorescence andphosphorescence. Fluorescent chemicals emit radiation due to the decayof the molecule which has been excited to a higher electronic state dueto the absorption of radiation. Phosphorescent molecules can emitradiation for a much longer time intervals. Detection of the specificwavelength of radiant energy emitted allows for the detection of targetswhich may be associated with the luminescent chemicals.

[0012] Bioluminescence is rapidly becoming a widely used method forlabeling many different types of compounds. Basically, a reducedsubstrate is reacted with oxygen and converted into an oxidized productwith an elevated or excited electronic state. The excited moleculedecays to the ground state and in the process, emits photons of light.This process has been found to occur in several strains of bacteria andfungi, in marine invertebrates such as sponges, and in shrimp andjellyfish. Bacteria which emit light are often found livingsymbiotically with fish in special luminescent organs. A wide variety ofterrestrial organisms such as earthworms, centipedes and insects alsopossess bioluminescent properties.

[0013] One group of compounds which undergo oxidation with the emissionof light are referred to as luciferins although their individualstructure may vary. The oxidized products are termed oxy-luciferins andthe enzymes which catalyze the process luciferases. The overall processis endothermic requiring chemical energy stored in one to two moleculesof adenosine triphosphate (ATP) per photon of light produced. Two typesof luciferase systems that have been widely used in molecular biologyare the bacterial system (Vibrio harveyi or V. fischeri) and the fireflysystem (Photinus pyralis).

[0014] Other labels which impart detectable properties to a substrateinclude chemicals with a unique absorption spectrum, electron spinresonance spectrum, optical activity, Raman spectrum or resonance Raman.spectrum Such labels are widely used in many fields including medicine,molecular biology and chemistry. For example, the visible or infraredabsorption spectrum of a molecule often constitutes a unique fingerprintwhich allows the molecule to be identified even in the presence of acomplex mixture. In the case of visible absorption, the molecule absorbsradiant energy over a specific wavelength range because of the presenceof an excited electronic state of the molecule whose energy oftransition from the ground state falls in the range 15-3 eV. In the caseof infrared absorption, bands are detected due to the excitation ofvibrational modes of the molecule. The frequency of these bands providesinformation about the presence or absence of characteristic moleculargroups such as disulfides, carbonyls and aromatic groups.

[0015] In another application of the spectroscopic properties ofmolecules, nuclear magnetic resonance (NMR) spectroscopy has beenextensively used to identify specific molecules in a mixture. The nucleiof atoms, such as protons in hydrogen atoms, which possess a netmagnetic moment, will align when placed into a magnetic field with thatfield and will precess about that field with a frequency (the Lamarfrequency) dependent on the individual properties of the particle. Todetermine the NMR spectrum, a sample of protons is placed within astrong magnetic field and irradiated with a range of radio frequenciesat a 90° angle with respect to the main field. This treatment causes allthe protons in the sample to absorb energy at their characteristicfrequency, flipping their magnetic orientations 90° with respect totheir original state. After the applied field is switched off, themolecules gradually relax to precess about the main field. Receivercoils which surround the sample detect the frequencies of precessingspins as a set of oscillating electric currents which constitute the NMRsignal.

[0016] All of these methods suffer from similar disadvantages. For themost part, targets do not have unique detectable properties such asinherent radioactivity or fluorescence. Labels must be attached whichare themselves detectable and therefore make the target detectable.However, the labeling process can result in a labeled product that is insome way permanently damaged. For example, fluorescent chemicals can beextremely toxic to cells. Long term exposure can result in a high degreeof cell death. Often, the labeling compound may have detrimental effectson a target's structure or activity. Protein structure is oftenadversely affected by the attachment of a detectable chemical moiety.Labeling of nucleic acids can interfere with their ability to betranslated, transcribed by polymerases or interact with DNA bindingproteins. In most cases, the chemical moiety must be removed. Further,the methods for removal of these chemical moieties which have detectablephysical properties often result directly in alteration of the moleculeor cell death.

[0017] There are a number of procedures, both complex and simple, whichhave been used to selectively detect and isolate target substrates. Oneprocedure which has revolutionized and greatly accelerated the detectionand identification of nucleic acids is polymerase chain reaction (PCR)technology. The principle concept of PCR is the rapid, large-scaleamplification of unique or even non-unique nucleic acid sequences inbiological samples. Using labeled primers with specific or randomsequences, the genetic code of very small quantities of nucleic acidscan be detected, amplified in number and subsequently characterizedthrough repetitive polymerization events. Although the nucleic acidsformed are new, the sequence of the original sample is maintained andcan be easily determined. As a nucleic acid sequence is the biological.code for the construction of virtually all proteins, the origin,evolutionary age, structure and composition of nearly any biologicalorganism or sample can be determined from knowledge of the sequence. Theprocedure has been proved useful in molecular and evolutionary biology,and has demonstrated applications in the detection, treatment andprevention of diseases and disorders in humans.

[0018] PCR technology, although revolutionary, carries with it the samelimitations as many conventional detection and isolation procedures.Label which has been incorporated into primers and ultimately newlyformed nucleic acids must be removed. This process, when possible, isfairly time consuming and often results is modification or destructionof the nucleic acid.

[0019] Another example of a process to render a substance specificallydetectable is to use binding molecules which have a particular affinityfor selected other molecules as occurs between binding of an antigen toan antigen-specific antibody. These chemical pairs, sometimes referredto as coupling agents, have been used extensively in detection andisolation procedures. Normally one of the molecules in this pair isimmobilized on an affinity medium such as used in chromatographicpacking material or a magnetic bead and used in the isolation of thetarget molecule. Some of the more useful coupling agents are biotin andavidin or the related protein, streptavidin. These agents have been usedin many separation techniques to facilitate isolation of one componentor another from complex mixtures

[0020] Biotin, a water-soluble vitamin, is used extensively inbiochemistry and molecular biology for a variety of purposes includingmacromolecular detection, purification and isolation, and incytochemical staining. Biotin also has important applications inmedicine in the areas of clinical diagnostic assays, tumor imaging anddrug delivery, and is used extensively in the field of affinitycytochemistry for the selective labeling of cells, subcellularstructures and proteins.

[0021] Biotin's utility stems from its ability to bind strongly to thetetrameric protein avidin, found in egg white and the tissues of birds,reptiles and amphibians, or to its chemical cousin, streptavidin,isolated from the bacterium Streptomyces. Typically, biotin or aderivative of biotin is first bound directly to a target molecule, suchas a protein or oligonucleotide, or to a probe using specific chemicallineage. The interaction of the linked biotin with either streptavidinor avidin conjugated to an affinity medium such as magnetic or sepharosebeads is then used in the isolation of the target molecule.Alternatively, the interaction of the covalently linked biotin withavidin or streptavidin conjugated to an enzyme such as horseradishperoxidase (HRP) which catalyzes a chromogenic reaction is used fordetection of the target molecule. Macromolecules that have been isolatedusing biotin-avidin technology are shown in Tables 1 and 2. TABLE 1Macromolecules Isolated by Direct Biotinylation Biotinylated TargetsElution Conditions References Membrane proteins acetate, pH 4 1, 2 andglycoproteins Antibodies low pH 3 Enzymes non-physiological 4 t-RNA 6 Mguanidine-HCl, 5 pH 2.5 rRNA 70% formic acid 6 nucleosomes SS-reductionof 7, 8 cleavable biotin DNA non-physiological 9

[0022] TABLE 2 Biological Materials Isolated Using Biotinylated BindingMolecules Binding Target Molecules Molecules Elution Conditions Refs.Glycoproteins conconavalin A 2% SDS 10 Membrane Antigens antibody SDS(boiling) 11 Estrogen Receptor estradiacetate, estradiol 12 InsulinReceptor insulin acetate, pH 5.0 13, 14 biotin Opoid Receptor enkephalinenkephalin 15 Human B antigen selection by FACS 16 lymphocytesLymphocyte monoclonal Mechanical 17, 18, 19 subpopulations antibodyagitation, erythrocyte lysis Plasmid DNA DNA 0.1 M NaOH 20 SpliceosomesRNA 90° C. in SDS 21 Recombinant DNA Cleavable biotin 22 Plasmids Heat,low ionic strength and phenol

[0023] While the utility of biotin continues to grow, there still existsmajor drawbacks in the use of biotin-streptavidin technology for manyapplications. This problem stems from the high affinity between biotinand streptavidin, precisely the molecular characteristic which makes itmost useful. Once a target molecule or cell is isolated through thestreptavidin-biotin interaction, release of the target molecule requiresdisruption of this interaction. Dissociation of biotin from streptavidinrequires very harsh conditions such as 6-8 molar (M) guanidinium-HCl, pH1.5. Such conditions also denature, and thereby inactivate, mostproteins and destroy most cells.

[0024] For example, a biotin derivative containing aN-hydroxysuccinimide ester group is commonly used to link biotin throughan amide bond to proteins and nucleic acids. Selective cleavage of thislinkage disrupts similar native chemical bonds in associated molecules.Biotin is also often used in the isolation of specific cells from aheterogeneous mixture of cells by binding a biotinylated antibodydirected against a characteristic cell surface antigen. The interactionof the biotinylated antibody with streptavidin-coated magnetic beads orsepharose particles can then be used effectively to isolate targetcells. Disruption of the antibody-antigen interaction normally requiresexposure of cells to conditions such as low pH or mechanical agitationwhich are adverse to the cell's survival. In general recovery of thetarget in a completely unmodified form is not possible.

[0025] Once the biotinylated DNA is bound to streptavidin it can only bereleased with extreme difficulty. Many diverse methods to remove thestreptavidin molecule have been suggested including digestion byproteinase K (M. Wilchek and E. A. Bayer, Anal. Biochem. 171:1, 1988).Proteinase K also digests nearby proteins and does a fairly poor job ofcompletely digesting the streptavidin. Significant amounts of thestreptavidin molecules remain attached, and further, removal ofstreptavidin does not release the biotin. Further, biotinylated DNAinterferes with subsequent use in a variety of methods includingtransformation of cells and hybridization based assays used fordetection of genetic diseases.

[0026] The essentially irreversible binding of biotin and streptavidinis also a serious limitation for the performance of multiple orsequential assays to detect a specific type of biomolecule,macromolecular complex, virus or cell present in a single sample.Normally, only a single assay can be performed because the enzymedetection system is streptavidin-based and streptavidin remains firmlybound to the biotinylated target or target probe. While differentchromogenic systems for detection are available, they are only oflimited applicability in situations where large numbers of probes areneeded.

[0027] An additional problem in the use of biotin-avidin technology isthe presence of endogenous biotin, either free or complexed to othermolecules, inside the sample to be purified or assayed. In this case,the endogenous biotin can result in the isolation or detection ofnon-target molecules. This can be a particularly severe problem in caseswhere a high signal-to-noise ratio is needed for accurate and sensitivedetection.

[0028] To remove biotin from an attached molecule, several chemicallycleavable biotin derivatives have been produced. ImmunopureNHS-SS-biotin (Pierce Chemical; Rockford, Ill.) consists of a biotinmolecule linked through a disulfide bond and an N-hydroxysuccinimideester group that reacts selectively with primary amines. Using thisgroup, NHS-SS-biotin can be linked to a protein and then the biotinportion removed by cleaving the disulfide bond with thiols. Thisapproach is of limited use since thiols normally disrupt nativedisulfide bonds in proteins. Furthermore, the cleavage still leaves thetarget cell or molecule modified since the spacer arm portion of thecomplex is not removed and the cleaving buffer must be eliminated fromthe sample.

[0029] One method for removal of biotin is the use of disulfide-basedcleavable biotins. However, the cleaved molecules possess a reactivesulfhydryl group which has a strong tendency to form disulfide bondswith other components of the mixture. Functional activity of thesesubstances containing sulfhydryl groups is severely compromised.Typically, activity of such protein is decreased or eliminated and suchnucleic acids will no longer hybridize rendering them useless forcloning. This method is also slow and requires the preparation ofcomplex solutions.

[0030] An additional limitation of biotin-avidin technology is thedifficulty of developing automated systems for the isolation and/ordetection of targets due to the problems of releasing the target fromthe biotin-avidin binding complex. This requires addition of specificchemical reagents and careful monitoring of the reactions.

[0031] Biotin-avidin technology has been combined with PCR techniquesfor the detection and isolation of nucleic acids and specific sequences.However, there still remains a fundamental problem which relates to thedifficulty of removing the incorporated biotin. This is normally notpossible using conventional biotins without irreversibly altering thestructure of the DNA. As discussed, biotinylation can interfere withsubsequent application of biotinylated probes as well as alter theproperties of the PCR product.

[0032] PCR products that contain biotinylated nucleotides or primerswhich are required for isolation cannot be used in conjunction withbiotinylated hybridization probes. The presence of biotin on the PCRproduct cause false signals from the avidin based enzyme-linkeddetection system. Biotin incorporation into DNA interferes with strandhybridization possibly due to the spacer arms linking the nucleotides tothe biotin molecules. Further, PCR products that are biotinylated arenot suitable material for cloning. PCR products which containbiotinylated nucleotides are difficult to analyze. Incorporation ofbiotinylated nucleotides into DNA causes a retardation of mobilityduring gel electrophoresis in agarose. This mobility shift renderscharacterization of PCR products difficult. As proper DNA-DNAhybridization is the basis for sensitive and accurate characterizationand sensitive assays, biotin-avidin binding systems are seriouslydisadvantaged.

[0033] Other coupling partners which can be used to detect and isolatetarget substances are cell adhesion molecules (CAMs). One of the wellcharacterized types is the endothelial cell adhesion molecule, LEC-CAM(leukocyte endothelial cell-cell adhesion molecule), now calledselectin. This molecule selectively binds to leukocytes. Its naturalfunction is to facilitate the transport of leukocytes through anendothelial layer of cells such as postcapillary venules to sites ofinflammation or tissue damage. There are many of these adhesionmolecules which have been identified in humans and other mammals thatrange in binding specificity from the very general to the highlyspecific. These include the endothelial cell adhesion ligands ICAM-1,VCAM-1 and ELAM-1, the β-integrins which consists of a family of threeproteins LFA-1, Mac-1, VLA-4, MO-1 and p150/95, carbohydrate bindingCAMs that appear on endothelial cells, platelets, and leukocytes, andthe cadherins, calcium dependent CAMs present on most cells. Attachmentof these molecules or the creation of fused proteins containing adhesiondomains can be used to facilitate isolation and detection of bindingpartners. However, once binding has occurred, complex, expensive andtime consuming biochemical manipulations and sometimes fairly harshchemical treatments are necessary to dissociate the molecules. Further,application of these molecules for general use is limited as bindingpartners must be located for each target of interest

[0034] Other coupling partners include nucleic acids and nucleic acidbinding proteins, lipids and lipid binding proteins, and proteins orspecific domains which have a particular affinity for each other. Thesecoupling partners suffer from similar drawbacks as the biotin-avidinsystem and the adhesion molecules.

[0035] Another fairly ubiquitous method of detection and isolation isgel electrophoresis. In this process, a uniform matrix or gel is formedof, for example, polyacrylamide, to which is applied an electric field.Mixtures applied to one end of the gel will migrate through the gelaccording to their size and interaction with the electric field.Mobility is dependent upon the unique characteristics of the substancesuch as conformation, size and charge. Mobilities can be influenced byaltering pore sizes of the gel, such as by formation of a concentrationor pH gradient, or by altering the composition of the buffer (pH, SDS,DOC, glycine, salt). One- and two-dimensional gel electrophoresis arefairly routine procedures in most research laboratories. Targetsubstances can be purified by passage through and/or physical extractionfrom the gel.

[0036] Methods for the detection and isolation of targets substancesalso include centrifugation techniques such asequilibrium-density-gradient centrifugation. This process is based onthe principal that under high centrifugal forces, stable gradients winbe established in salt solutions. Mixtures subjected to high speedcentrifugation will segregate individual. components according to theirspecific densities. Although useful, all of these procedures are morequantitative than qualitative.

[0037] A major advance in detection and isolation methodology was theadvent of liquid chromatography. Chromatography, and in particularcolumn chromatography, comprises some of the most effective and flexiblepurification methods available. Common to most procedures is the use ofopen cylinders containing a hydrated matrix material. Some of thetypical matrix materials which are presently used, for example, in gelfiltration, affinity chromatography and ion exchange chromatography,include sepharose (bead formed gel prepared from agarose: PharmaciaBiotech; Piscataway, N.J.), sephadex (a bead-formed gel prepared bycross-linking dextran with epichlorohydrin: Pharmacia Biotech;Piscataway, N.J.) and sephacryl (covalently cross-linked allyl dextranwith N,N′-methylene bisacrylamide: Pharmacia Biotech; Piscataway, N.J.).Basically, a heterogenous sample or mixture is applied to the top of thecolumn followed with a suitable buffer. Substances within the mixturedisplay differential migration through the column in relation to othermaterials within the sample and is collected in fractions at the otherend of the column Fractions are individually analyzed for the presenceof target and positive fractions pooled.

[0038] Alternatively, target in the sample may selectively bind to thecolumn material in the presence of buffer a process known as affinitychromatography. After binding, unbound material is removed bycontinuously washing the column with buffer. Target molecules aresubsequently released from the column by application of an elutionbuffer which causes dissociation. Fractions are collected as they eluteoff of the column and collected. In gel-exclusion chromatography, across-linked dextran is utilized as column matrix material.Cross-linking can be varied to alter the effective pore size of thecolumn material and the dextran can be coupled to a wide variety ofchemical moieties to selectively capture target. Ion-exchangechromatography takes advantage of the fact that targets, for exampleproteins, can differ enormously in their affinity for positive ornegative charges on column materials. The affinity of a material for atarget is proportional to the salt concentration of the buffer. Byraising or lowering the salt concentration, it is possible to changeaffinity of target to column material.

[0039] Affinity column chromatography makes use of chemical groups thathave special attraction to the targets of interest. For example, enzymespreferentially bind to certain naturally associated cofactors. Columnmaterials with attached cofactors will selectively bind to such targetenzymes. Enzyme purification becomes a relatively simple andstraightforward matter. In a similar fashion, enzyme-specific antibodiescan be coupled either covalently or non-covalently to a column matrix.The unique affinity of an antibody for its target antigen allows for theselective removal of target from a heterologous mixture of substances.Detection and isolation is again a fairly simple matter.

[0040] Two relatively well-established procedures, high-performanceliquid chromatography (HPLC) including reverse-phase HPLC andsize-exclusion HPLC, and the more recent technique fast-performanceliquid chromatography (FPLC) which can handle larger sample volumes thanHPLC, is based on standard chromatographic techniques, but usingextremely high pressures (5,000 to 10,000 psi and more). Due to thehigher pressures, finer column materials can be utilized and separationscan be performed faster and with better resolution.

[0041] Although chromatography is an invaluable tool it too has itslimits. Materials to be separated must be solubilized into a suitablebuffer which will not adversely affect the column. Further, substratemixtures and targets must be capable of passing through a column matrixin a reasonable period of time. Although HPLC can sometimes shorten thistime period, only small quantities can be detected and the highpressures can damage isolated column material.

SUMMARY OF THE INVENTION

[0042] The present invention overcomes the problems and disadvantagesassociated with current strategies and designs and provides new methodsfor the detection and isolation of molecules from complex mixtures.

[0043] One embodiment of the invention is directed to bioreactive agentscomprising a detectable moiety bonded to a photoreactive moiety whereinthe photoreactive moiety contains at least one group capable ofcovalently bonding to a substrate to form a conjugate that can beselectively photocleaved to release said substrate. Detectable moietiesshould have a selectively detectable physical property such asfluorescence, absorption or an ability to specifically bind to acoupling agent such as avidin or streptavidin, antibodies, antigens orbinding proteins. The photoreactive moiety should be capable of formingone or more covalent bonds with a chemical group of a substrate. Thosecovalent bonds may be cleaved or photocleaved with the electromagneticradiation releasing the substrate.

[0044] The bioreactive agent may have a chemical structure selected fromthe group consisting of:

[0045] wherein X is selected from the group consisting of a halogen, N₂,CH₂-halogen, —N═C═O, —N═C═S, —S—S—R, NC₂H₄, —NC₄H₂O₂, —OH, —NHNH₂,—OP(OR₃)N(R₄)R₅ and —OCO—G, wherein G is selected from the groupconsisting of a halogen, N₃, O-esters and N-amides; R₁, R₂, R₃, R₄ andR₅ are selected from the group consisting of hydrogen, alkyls,substituted alkyls, aryls and substituted aryls, —CF₃, —NO₂, —COOH and—COOR, and may be the same or different; A is a divalent functionalgroup selected from the group consisting of —O—, —S— and —NR₁; Ycomprises one or more polyatomic groups which may be the same ordifferent; V comprises one or more optional monoatomic groups which maybe the same or different; Q comprises an optional spacer moiety; m1 andm2 are integers from 0-5 and may be the same or different; and Dcomprises a detectable moiety which is distinct from R₁-R₅.

[0046] Another embodiment of the invention is directed to conjugatescomprising a bioreactive agent photocleavably coupled to a substratewherein said agent comprises a detectable moiety bonded to aphotoreactive moiety, wherein said conjugate can be selectively cleavedwith electromagnetic radiation to release said substrate. Suitablesubstrates which can be coupled to the bioreactive agent includeproteins, peptides, amino acids, amino acid analogs, nucleic acids,nucleosides, nucleotides, lipids, vesicles, detergent micells, cells,virus particles, fatty acids, saccharides, polysaccharides, inorganicmolecules, metals, and derivatives and combinations thereof Substratesmay be pharmaceutical agents such as cytokines, immune systemmodulators, agents of the hematopoietic system, chemotherapeutic agents,radio-isotopes, antigens, anti-neoplastic agents, recombinant proteins,enzymes, PCR products, receptors, hormones, vaccines, haptens, toxins,antibiotics, nascent proteins, cells, synthetic pharmaceuticals andderivatives and combinations thereof.

[0047] Conjugates may have a chemical structure selected from the groupconsisting of:

[0048] wherein SUB comprises a substrate; R₁ and R₂ are selected fromthe group consisting of hydrogen, alkyls, substituted alkyls, aryls,substituted aryls, —CF₃, —NO₂, —COOH and —COOR, and may be the same ordifferent; A is a divalent functional group selected from the groupconsisting of —O—, —S— and —NR₁; Y comprises one or more polyatomicgroups which may be the same or different; V comprises one or moreoptional monoatomic groups which may be the same or different; Qcomprises an optional spacer moiety; m1 and m2 are integers between 1-5which can be the same or different; and D comprises a detectable moietywhich is distinct from R₁ and R₂.

[0049] Another embodiment of the invention is directed to pharmaceuticalcompositions comprising the conjugate plus a pharmaceutically acceptablecarrier such as water, an oil, a lipid, a saccharide, a polysaccharide,glycerol, a collagen or a combination thereof. Pharmaceuticals may beused in the prophylaxis and treatments of diseases and disorders inhumans and other mammals.

[0050] Another embodiment of the invention is directed to methods forisolating targets from a heterologous mixture. Briefly, a conjugate iscreated by coupling a bioreactive agent to a substrate by a covalentbond which is selectively cleavable with electromagnetic radiationwherein the bioreactive agent is comprised of a photoreactive moietybonded to a detectable moiety. The conjugate is contacted to theheterologous mixture to couple substrate to one or more targets. Thecoupled conjugate is separated from the mixture and treated withelectromagnetic radiation to release the substrate, and the targetsisolated. This method can be used to isolate targets such as immunesystem modulators, cytokines, agents of the hematopoietic system,proteins, hormones, gene products, antigens, cells, toxins, bacteria,membrane vesicles, virus particles, and combinations thereof fromheterologous mixtures such as biological samples, proteinaceouscompositions, nucleic acids, biomass, immortalized cell cultures,primary cell cultures, vesicles, animal models, mammals, cellular andcell membrane extracts, cells in vivo and combinations thereof

[0051] Another embodiment of the invention is directed to targetmolecules isolated by the above methods which may be used inpharmaceutical compositions or other compositions and mixtures forindustrial applications.

[0052] Another embodiment of the invention is directed to methods forisolating targets from a heterologous mixture. A conjugate is createdcomprising a bioreactive agent coupled to a substrate by a covalent bondwhich is selectively cleavable with electromagnetic radiation, whereinsaid bioreactive agent is comprised of a photoreactive moiety bonded toa detectable moiety and the substrate is a precursor of the target. Theconjugate is contacted with the heterologous mixture to incorporatesubstrate into targets. The incorporated conjugate is separated from themixture, treated with electromagnetic radiation to release thesubstrate, and the targets isolated. This method is useful for thedetection and isolation of nascent proteins, nucleic acids and otherbiological substances.

[0053] Another embodiment of the invention is directed to methods forisolating targets from a heterologous mixture. A conjugate is createdwhich is comprised of a bioreactive agent coupled to a receptor by acovalent bond which is selectively cleavable with electromagneticradiation, wherein said bioreactive agent is comprised of aphotoreactive moiety bonded to a detectable moiety. The conjugate iscontacted with the heterologous mixture to couple receptor to targetsand the coupled receptor-targets separated from the mixture. Theseparated conjugate is treated with electromagnetic radiation to releasethe receptor and the targets isolated.

[0054] Another embodiment of the invention is directed to methods forisolating target cells from a heterologous mixture. A conjugate iscreated comprising a bioreactive agent coupled to a cell receptor by acovalent bond which is selectively cleavable with electromagneticradiation, wherein the bioreactive agent is comprised of a photoreactivemoiety bonded to a detectable moiety. The conjugate is contacted withthe heterologous mixture to couple receptor to target cells. The coupledconjugate is separated from the mixture and treated with electromagneticradiation to release the substrate. Target cells are then easilyisolated such as by automation.

[0055] Another embodiment of the invention is directed to methods forcreating a photocleavable oligonucleotide. A conjugate is createdcomprising a bioreactive agent coupled to a phosphoramidite which may bea purine-phosphoramidite or a pyrimidine-phosphoramidite. Theoligonucleotide is synthesized using photocleavable phosphoramidites.The process can be performed manually or automated to be carried out byan oligonucleotide synthesizer.

[0056] Another embodiment of the invention is directed to methods fordetermining an in vivo half-life of a pharmaceutical in a patient. Aconjugate is formed by coupling the pharmaceutical to a bioreactiveagent with a covalent bond that can be selectively cleaved withelectromagnetic radiation, wherein said bioreactive agent comprises aphotoreactive moiety bonded to a detectable moiety. The conjugate isadministered to the patient and at least two or more biological samplesare removed from the patient at various times after administration ofthe conjugate. The samples are treated with electromagnetic radiation torelease the pharmaceutical from the bioreactive agent and the amount ofthe bioreactive agent in the biological samples determined. The in vivohalf-life of the pharmaceutical can be determined.

[0057] Another embodiment of the invention is directed to methods forthe controlled release of a substrate into a medium. A conjugatecomprised of a bioreactive agent coupled to the substrate by a covalentbond which can be selectively cleaved with electromagnetic radiation iscreated wherein the bioreactive agent is comprised of a detectablemoiety bonded to a photoreactive moiety. The conjugate is bound to asurface of an article which is placed into the medium. The surface ofthe article is exposed to a measured amount of electromagnetic radiationfor the controlled release of the substrate into the medium.

[0058] Another embodiment of the invention is directed to methods fordetecting a target molecule in a heterologous mixture. A conjugate isformed by coupling a substrate to a bioreactive agent with a covalentbond that is selectively cleavable with electromagnetic radiation,wherein the bioreactive agent is comprised of a detectable moiety bondedto a photoreactive moiety. The conjugate is contacted with theheterologous mixture to couple substrate to one or more targetmolecules. Uncoupled conjugates are removed and the coupled conjugatesare treated with electromagnetic radiation to release the detectablemoiety. The released detectable moiety can now be easily detected.

[0059] Another embodiment of the invention is directed to methods fordetecting a target molecule in a heterologous mixture. A conjugate,comprising a substrate coupled to a bioreactive agent, is formed andcontacted with a heterologous mixture to couple a conjugate to one ormore target molecules. Uncoupled conjugates are removed and the coupledconjugates are treated with electromagnetic radiation to releasesubstrate. Released substrate is detected and can be further isolated.

[0060] Another embodiment of the invention is directed to methods forthe isolation of a PCR product. A bioreactive agent is conjugated to oneor more oligonucleotide primers with a covalent bond that is selectivelycleavable with electromagnetic radiation, wherein the bioreactive agentis comprised of a detectable moiety bonded to a photoreactive moiety. Anucleic acid sequence is PCR amplified with the conjugated primers. Theamplified sequences are isolated and subsequently treated withelectromagnetic radiation to release the bioreactive agent.

[0061] Another embodiment of the invention is directed to methods fortreating a disorder by the controlled release of a therapeutic agent ata selected site. A conjugate is formed by bonding a bioreactive agent tothe therapeutic agent with a bond that is selectively cleavable withelectromagnetic radiation, wherein the bioreactive agent is comprised ofa directable moiety bonded to a photoreactive moiety wherein thedirectable moiety has an affinity for the selected site. The conjugateis administered to a patient having the disorder. The selected site issubjected to a measured amount of electromagnetic radiation for thecontrolled release of the therapeutic agent to treat the disorder.

[0062] Another embodiment of the invention is directed to kits fordetecting a disorder in biological samples containing conjugatescomprised of a bioreactive agent covalently bonded to a diagnostic agenthaving an affinity for an indicator of the disorder in the biologicalsample, wherein the covalent bond is selectively cleavable withelectromagnetic radiation.

[0063] Another embodiment of the invention is directed to kitscomprising a bioreactive agent covalently bonded to an oligonucleotide.The photocleavable oligonucleotide may be double-stranded orsingle-stranded and may possess restriction enzyme recognition sitesuseful in cloning and other procedures in molecular biology.

[0064] Other embodiments and advantages of the invention are set forth,in part, in the description which follows and, in part, will be obviousfrom this description or may be learned from the practice of theinvention.

DESCRIPTION OF THE FIGURES

[0065]FIG. 1 Examples of photocleavable agents.

[0066]FIG. 2 Photocleavable biotins with various photoreactive moieties.

[0067]FIG. 3 Schematic representation of photocleavable biotin

[0068]FIG. 4 Mechanism of photocleavage in 2-nitrobenzyl-based systems.

[0069]FIG. 5 Synthesis of photocleavable biotin.

[0070]FIG. 6 Chemical variations of photocleavable agents.

[0071]FIG. 7 Photolysis of PCBs.

[0072]FIG. 8 PCB conjugates.

[0073]FIG. 9 Possible amino acid linkages of PCB.

[0074]FIG. 10 (A) Aminoacylation of tRNA, and (B) a comparison betweenenzymatic 5 and chemical aminoacylation.

[0075]FIG. 11 The four basic steps in the isolation of pure substrateusing PCB.

[0076]FIG. 12 Method for the detection of target protein and itsantibody using PCB.

[0077]FIG. 13 PCB-phosphoramidites and PCB-nucleotides.

[0078]FIG. 14 Two methods (A and B) for the isolation of PCR-productusing PCB.

[0079]FIG. 15 Comparison of methods for the construction of a cDNAlibrary (A) with PCB and (B) without PCB. SEQ. ID. No.: 1

[0080]FIG. 16 PCB lipids.

[0081]FIG. 17 Immunoselective cell separation using PCB.

[0082]FIG. 18 Synthesis of photocleavable biotin-NHS ester, compound 18.

[0083]FIG. 19 Synthesis of photocleavable biotin-NHS ester, compound 25.

[0084]FIG. 20 Method for sequential ELISA using PCB.

[0085]FIG. 21 Synthesis of photocleavable coumarin.

DESCRIPTION OF THE INVENTION

[0086] As embodied and broadly described herein, the present inventionis directed to agents and conjugates used in the detection and isolationof targets such as chemicals, macromolecules, cells and any identifiablesubstance from a mixture. Agents comprise a detectable moiety bound to aphotoreactive moiety. Conjugates comprise agents which are coupled tosubstrates by one or more covalent bonds which, by the presence of thephotoreactive moiety, are selectively cleavable with electromagneticradiation. The invention is also directed to methods for the isolationand detection of targets using these agents and conjugates, to kitswhich utilize these methods for the detection of diseases and disordersin patients, and to methods for the detection and isolation of nearlyany substance from a heterologous mixture.

[0087] There are many methods currently available for the detection andisolation of a desired substance or target from a complex mixture. Mostof these methods require the specific labeling of the substance ortarget to be detected, detection of that label and subsequent removal oflabel from target. Although straightforward, current detection andisolation methodologies possess a number of problems. For example, it isoften difficult to specifically attach target with label. Affinity oflabel for target may be low, suitable points of attachment may not beavailable on specific substances and the label and the target may simplybe chemically or physically incompatible. In addition, label may hinderor completely destroy the functional activity of the target frustratingthe purpose of isolation. Isolated targets are unavoidably contaminatedor inactivated due to the presence of a toxic or damaging label. Atypical example of this sort of problem is the isolation of cells boundwith biotin after selection by coupling to streptavidin. The powerfulaffinity of biotin for streptavidin makes the isolation procedurerelatively straightforward and specific, however, the isolated cells areoften dead and dying due to the toxic effects of the coupling agents orthe harsh isolation procedures. This is also true when attempting toisolate active proteins from biological samples and other complexmixtures for later use. The presence of label may denature or render theprotein product inactive or simply unacceptable for in vivo use undercurrent FDA standards and guidelines. Removal of the agent sometimesovercomes these problems, however, methods to separate and remove labelfrom target are generally rather harsh, take a significant amount oftime, effort and expense, and, for the most part, result in fairly lowyields of the final product. Viability and functional activity of thetarget is often severely impaired and is often destroyed.

[0088] The invention overcomes these problems by providing detectable,bioreactive agents which can detect and isolate targets. Agents of theinvention comprise a detectable moiety and a photoreactive moiety, andcan be covalently coupled to a variety of target substrates. A covalentbond between agent and substrate can be created from a wide variety ofchemical moieties including amines, hydroxyls, imidazoles, aldehydes,carboxylic acids, esters and thiols. Agent-substrate combinations arereferred to herein as conjugates. Through the presence of the detectablemoiety, conjugates can be quickly and accurately detected and targetisolated. Further, these conjugates are selectively cleavable whichprovides unique advantages in isolation procedures. Substrate can beseparated from agent quickly and efficiently. Complex technicalprocedures and highly trained experts are not required. New attachmentand separation procedures do not need to be developed for every newtarget to be isolated. Following isolation, it is a relatively simplematter to treat the conjugate with electromagnetic radiation and releasethe substrate. Released substrate is preferably functionally active andstructurally unaltered. Nevertheless, minor chemical alterations in thestructure may occur depending on the point of attachment. It isgenerally preferred that such alterations not effect functionalactivity. However, when functional activity does not need to bepreserved, such changes are of no considerations and may even be usefulto identify and distinguish targets isolated by methods of theinvention.

[0089] Targets, as referred to herein, are those substances beingidentified, characterized or isolated using the agents, conjugates andmethods of the invention. Substrates, as referred to herein, are thosesubstances which are covalently attached to the bioreactive agent.Substrates may also be referred to as targets when the target beingidentified specifically binds to the bioreactive agent.

[0090] One embodiment of the invention is directed to a bioreactiveagent comprising a detectable moiety bonded to a photoreactive moietywherein the photoreactive moiety contains at least one group capable ofcovalently bonding to a substrate to form a conjugate. The resultingconjugate can be selectively cleaved to release said substrate or,alternatively, to release any chemical group or agent of the conjugate.Cleavage, as referred to herein, is by photocleavage or a cleavage eventtriggered by the application of radiation to the conjugate. Theradiation applied may comprise one or more wavelengths from theelectromagnetic spectrum including x-rays (about 0.1 nm to about 10.0nm; or about 10¹⁸ to about 10¹⁶ Hz), ultraviolet (UV) rays (about 10.0nm to about 380 nm; or about 8×10¹⁶ to about 10¹⁵ Hz), visible light(about 380 nm to about 750 nm; or about 8×10¹⁴ to about 4×10¹⁴ Hz),infrared light (about 750 nm to about 0.1 cm; or about 4×10¹⁴ to about5×10¹¹ Hz), microwaves (about 0.1 cm to about 100 cm; or about 10⁸ toabout 5×10¹¹ Hz), and radio waves (about 100 cm to about 10⁴ m; or about10⁴ to about 10⁸ Hz). Multiple forms of radiation may also be appliedsimultaneously, in combination or coordinated in a step-wise fashion.Radiation exposure may be constant over a period of seconds, minutes orhours, or varied with pulses at predetermined intervals.

[0091] Typically, the radiation source is placed at a specified distancefrom the conjugate to be irradiated. That distance may be empiricallydetermined or calculated from the energy loss produced between thesource and the target and the amount of energy emitted by the source.Conjugate may be in solution or attached to a solid support which may bea type of glass, ceramic, polymer or semiconductor surfaces. Typicalsolid supports are nitrocellulose membranes, agarose beads, magneticbeads coated with streptavidin, semiconductor surfaces and resins.Preferably, the radiation applied is UV, visible or IR radiation of thewavelength between about 200 nm to about 1,000 nm, more preferablybetween about 260 nm to about 600 nm, and more preferably between about300 nm to about 500 nm. Radiation is administered continuously or aspulses for hours, minutes or seconds, and preferably for the shortestamount of time possible to minimize any risk of damage to the substrateand for convenience. Radiation may be administered for less than aboutone hour, preferably less for than about 30 minutes, more preferably forless than about ten minutes, and still more preferably for less thanabout one minute. Visible, UV and IR radiation are also preferred as allthree of these forms of radiation can be conveniently and inexpensivelygenerated from commercially available sources.

[0092] The power density or intensity of light per area necessary toselectively cleave the covalent bond is very small which makes thephotocleavable process practical. Maximization of efficiency alsominimizes exposure time necessary to achieve selective cleavage andprovide a minimum of undesirable background effects.

[0093] One part of the bioreactive agent is the detectable moiety. Thedetectable moiety is a chemical group, structure or compound thatpossesses a specifically identifiable physical property which can bedistinguished from the physical properties of other chemicals present inthe heterologous mixture. Fluorescence, phosphorescence and luminescenceincluding electroluminescence, chemiluminescence and bioluminescence areall detectable physical properties not found in most substances, butknown to occur or to be inducible in others. For example, reactivederivatives of dansyl, coumarins, rhodamine and fluorescein are allinherently fluorescent when excited with light of a specific wavelengthand can be specifically bound or attached to other substances. Coumarinhas a high fluorescent quantum yield, higher than even a dansyl moiety,and facilitates detection where very low levels of target that are beingsought. Coumarin is structurally similar to tryptophan, which can beuseful in for example in the translation of nascent proteins withnon-native amino acids. It may also be useful to combine certaindetectable moieties to facilitate detection or isolation. Preferably thedetectable moiety is a fluorescent compound and the preferredfluorescent compounds are listed in Table 3, all of which arecommercially available (Sigma Chemical; St. Louis, Mo.). TABLE 3Fluorescent Labeling Compounds4-acetamido-4′-isothiocyanatostilbene-2-2′-disulfonic acid7-amino-4-methylcoumarin (AMC) 7-amino-4-trifluoromethylcoumarinN-(4-anilino-1-naphthyl) maleimide 4′,6-diamidino-2-phenylindole (DAPI)5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF)4,4′-dilsothiocyanatostilbene-2,2′-disulfonic acid tetramethylrhodamineisothiocyanate (TRITC) quinolizino fluorescein isothiocyanate (QFITC)dansyl chloride eosin isothiocyanate erythrosin B fluorescaminefluorescene fluorescein derivatives 4-methylumbelliferoneo-phthaldialdehyde rhodamine B rhodamine B derivatives rhodamine 6Grhodamine 123 sulforhodamine B sulforhodamine 101 sulforhodamine 101acid chloride

[0094] Luminescence can also be induced in certain chemicals referred toas luciferins. Energetic molecules such as ATP supply chemical energyfor catalytic activities of luciferase enzymes causing the luciferins toemit light. Reagents for both the bacterial luciferase system (Vibrioharveyi or V. fischeri) and the firefly luciferase system (Photinuspyralis) are available from a variety of commercial sources (e.g. SigmaChem. Co; St. Louis, Mo.).

[0095] Preferably, the luminescent agent has a high quantum yield offluorescence at a wavelength of excitation different from that used toperform the photocleavage. Upon excitation at such wavelengths, theagent is detectable at low concentrations either visually or usingconventional luminescence detectors and fluorescence spectrometers.Electroluminescence, produced by agents such as ruthenium chelates andtheir derivatives, or agents that possess nitroxide moieties and similarderivatives are preferred when extreme sensitivity is desired (J.DiCesare et al., BioTechniques 15:152-59, 1993). These agents aredetectable at the femtomolar ranges and below.

[0096] Application of an electric field will also induce a detectableresponse in certain chemicals' due to a net electric charge whichinduces the substance to migrate in an electric field. Magnetism mayalso be a detectable property if a magnetized substance such as iron oranother magnetized metal is or is associated with the detectable moiety.

[0097] Other forms of detectable physical properties include anidentifiable electrical polarizability, electron spin resonance andRaman scattering. Agents may also undergo a chemical, biochemical,enzymatic, electrochemical or photochemical reaction such as a colorchange in response to external electromagnetic fields or theintroduction of other substances. Such electromagnetic fields andsubstances may be a catalyst or another reactant molecule that allowsfor detection of the bioreactive agent or transforms the agent into adetectable moiety.

[0098] All of these labeling agents can be specifically detected usingthe appropriate detector or detection system such as a spectrometer orelectrophoretic or chromatographic systems. At times, it may bepreferable to have a visually discernable detection system such as onethat will trigger a photoelectric cell or one that can be detected andrecorded manually. Spectrometers including absorption and fluorescencespectrometers are very sensitive detectors of specific energy ofabsorptions or emissions from many detectable moieties. Detection andsorting of target may be automated as in the case of fluorescenceactivated cell sorters (FACS) which detect and isolate cells thatpossess a fluorescent label. Targets may be detected and sorted manuallyas can be done quite simply with magnetized conjugates using a magnet.

[0099] Additional physical properties which can be easily and accuratelydetected include chromaticism (e.g. violet=about 400-430 nm, blue=about450-500 nm, green=about 550 nm, yellow=about 600 nm, orange=about 650nm, red=about 700-750 nm), electromagnetic absorbance, enzyme activityor the ability to specifically bind with a coupling agent. Usefulcoupling agents include biotin, avidin, streptavidin, nucleic acids,nucleic acid and lipid binding proteins, haptens, antibodies, receptors,carbohydrates, immunogenic molecules, and derivatives and combinationsthereof. The detectable moiety may have a combination of theseproperties allowing its selection from a wide variety of backgroundmaterials. Some examples of the chemical structures of photocleavableagents of the invention are depicted in FIG. 1.

[0100] Another preferred detectable moiety is a coupling agent and thepreferred coupling agent is biotin or a biotin derivative.Biotin-containing bioreactive agents are referred to herein asphotocleavable biotins or PCBs. The binding between the egg-whiteprotein avidin, a tetrameric protein found in avian eggs with the watersoluble vitamin, biotin, is one of the strongest interactions known inbiology having an association constant (K_(a)) of about 10¹⁵M⁻¹,exceeding that of antibody-antigen interactions (M. Wilchek and E. A.Bayer, Methods Enzymol. 184, 1990). The bacterial counterpart to avidinis streptavidin, found in Streptomyces avidinii, which is slightly morespecific for biotin than avidin. This strong interaction, along with theability to covalently link biotin to a variety of substrates includingproteins, nucleic acids, lipids, and receptor ligands such asneuropeptides and hormones, has resulted in a vast array of uses forthese coupling agents all of which can be improved or enhanced with theuse of PCB.

[0101] A wide variety of biotinyl moieties can be used to form a PCBmolecule. Biotin (C₁₀H₁₆N₂O₃S) has a molecular weight of 244.31 daltonsand is comprised of a ring linked to an alkyl chain terminated by acarboxyl group. Numerous modifications can be made to the biotin moietywhich involve changes in the ring, spacer arm and terminating group, allof which still exhibit a high affinity for streptavidin, avidin andtheir derivatives. Examples of photocleavable biotins that can bedesigned based on various photoreactive moieties are depicted in FIG. 2.

[0102] The detection and isolation of chemical, biochemical andbiological materials using the interaction between biotin andstreptavidin is normally based on the immobilization of avidin orstreptavidin to a surface (e.g. membranes, gels, filters, microtiterwells, magnetic beads). To that surface is applied a solution containingbiotin coupled to targets which then bind to the streptavidin-coatedsurface. Biotin-containing target molecules can be isolated andnon-biotinylated components washed away. Alternatively, biotinylatedtarget molecules can be separated from a heterogeneous mixture usingstreptavidin-containing affinity columns. Biotinylated macromoleculesincluding nucleic acids (DNA or RNA), proteins and protein-containingcomplexes, and even cells whose surface has been biotinylated or boundto a biotinylated antibody can be detected and isolated with thesetechniques.

[0103] As stated, biotin can be coupled to a wide variety of moleculesincluding proteins, carbohydrates and nucleic acids. The availability ofbiotin derivatives has expanded this range even further. For example,biotin derivatives have been prepared with functionalities which arereactive towards amines, phenols, imidazoles, aldehydes, carboxylicacids and thiols. Biotin can also be incorporated into proteins, DNA andRNA by first attaching the biotin to building blocks of macromoleculessuch as amino acid or nucleotides which can be directly attached tothese molecules or incorporated during their synthesis by chemical orenzymatic means.

[0104] Unlike conventional biotins, photocleavable biotins enable one torelease or elute the bound substrate from the immobilized avidin,streptavidin or their derivatives in a completely unmodified form. Thisis extremely useful and an important improvement over existing biotinsfor a number of reasons. Biotinylation of the target material can impedeits subsequent use or characterization. Biotinylation of a protein canalter its activity, electrophoretic mobility, ability to bind asubstrate, antigenicity, ability to reconstitute into a native form andability to form multisubunit complexes. In contrast, usingphotocleavable biotin, once the biotin is photocleaved from the proteinor protein/binding complex, all the native properties and function willbe restored to its native form for further use and characterization.Listed in Table 4 are some of the substrates to which a photocleavableagent such as PCB can be linked. TABLE 4 Chemical linkage ofPhotocleavable Biotins with different molecules Resulting linkageMolecule or Functional Group Reactive moiety and reaction Assemblage onthe Molecule on the PCB conditions Amino acids, Amino group or NHS-esterAmide linkage proteins, enzymes R—NH₂ or antibodies Amino acids, R—OHchloroformate Ester linkage proteins, enzymes carboxylic acid orantibodies Amino acids, R—COOH Reaction with Ester linkage proteins,enzymes parent alcohol or antibodies (DCC coupling) Nucleotides,Aromatic amines chloroformate Amides RNA or DNA molecules CarbohydratesSugar hydroxy chloroformate Ester linkage RNA for R—OH ribonucleotidesNucleotide Phosphate groups diazoethane Phosphate ester phosphoramiditesLipids/ R—NH₂ Chloroformate amide Phosphatidyl NHS-ester serineCarbohydrates Sugar hydroxyl Chloroformate Ester linkage

[0105] There are a number of chemical moieties available in bioreactiveagents and conjugates of the invention. For example, an NHS-esterfunctionality introduced in PCB is highly reactive and can selectivelyreact with aliphatic amino groups that are present in proteins. Anotherexample is a phosphoramidite moiety which is highly reactive and canselectively react with hydroxyl groups of nucleic acids. In cases wherechemical moieties like carboxyl (—COOH) or phosphate groups needmodification, a precursor of PCB in the form of the parent alcohol canbe used to form appropriate ester-type linkages. These derivatives canbe chemically linked to a variety of macromolecules and molecularcomponents including amino acids, nucleotides, proteins andpolypeptides, nucleic acids (DNA, RNA, PNA), lipids, hormones andmolecules which function as ligands for receptors.

[0106] The application of biotin-avidin technology for the detection andisolation of chemical and biological materials has also been broadenedby the use of binding molecules which are first biotinylated and thenallowed to selectively interact with the target molecule to be isolated.Isolation of the target molecule or cell is facilitated by the bindingof the biotinylated binding-complex to the streptavidin-containingcolumn or streptavidin-coated magnetic beads. Binding molecules includeantibodies which selectively bind to specific antigens, DNA probes whichselectively bind to specific DNA sequences and ligands which selectivelybind to specific receptors. This approach has been used to isolate awide variety of macromolecules and cells (Tables 2 and 3). However, suchisolation methods require that the biotinylated target be released fromthe bound streptavidin. Disruption of this bond typically requiresnon-physiological conditions such as low pH and high concentration ofguanidinium-HCl which is usually damaging for the target molecule orcell. Even after disruption of the streptavidin-biotin interaction, thetarget or binding molecule remains partially or completely biotinylatedwhich can interfere with later uses. Further, elution conditions arenon-physiological and can also be disruptive to the target molecule orcell. In contrast, using photocleavable biotins substrate can be quicklyand easily cleaved from biotin with little to no effect on substrateconformation or activity.

[0107] The use of PCB in any of the usual detection and purificationprocedures, including those discussed above, represents a significantsavings of time, energy and ultimately cost. In addition, a variety ofderivatives of avidin and streptavidin are commercially available whichhave been modified through chemical or genetic means. These samederivatives can be used with PCB. One example is ImmunoPure NeutrAvidinsold commercially (Pierce Chemical; Rockford, Ill). This protein is amodified avidin derivative which has been deglycosylated and does notcontain the RYD domain that serves as a universal recognition sequencefor many cell receptors. Non-specific adsorption to other proteins andcell surfaces is greatly reduced.

[0108] The major molecular elements of a photocleavable biotin (PCB) area photoreactive moiety and a biotinyl moiety which constitutes thedetectable moiety (FIG. 3). The photoreactive moiety and the biotinylmoiety are linked together with a spacer arm to form the PCB molecule.The photoreactive moiety contains a five or six membered ringderivatized with functionalities represented by X, Y and A—C(O)—G,wherein X allows linkage of PCB to the biomolecular substrate. In thepreferred embodiment, Y represents a substitution pattern on a phenylring containing one or more substituents such as nitro or alkoxy groups.The functionality W represents the group that allows linkage of thecross-linker moiety to the photoreactive moiety. The purpose of thespacer arm is to increase the access of the biotin moiety for effectiveinteraction with streptavidin, and thus, increase the bindingefficiency. Typically these can be constructed using either long alkylchains or using multiple repeat units of caproyl moieties linked viaamide linkages.

[0109] Choice of photolabile group, spacer arm and the biotinyl moietydepends on the target substrate including amino acids, proteins,antibodies, nucleotides, DNA or RNA, lipids, carbohydrates and cells towhich the photocleavable biotin is. to be attached. It also depends onthe exact conditions for photocleavage and the desired interactionbetween the biotinyl moiety and streptavidin, avidin or theirderivatives. Some of the various choices for the photolabile group andlinker arms for PCB are shown in FIG. 2.

[0110] Additional types of coupling agents include antibodies, antibodyfragments and antigens. Antibodies have the advantage that they can bindto their respective antigen with great specificity. Substrates which areantigens can be detected by their ability to specifically bind toavailable antibodies or to antibodies which can be easily created.Useful antibodies or antibody fragments may be monoclonal or polyclonaland are preferable of the class IgG, but may also be IgM, IgA, IgD orIgE. Other preferred detectable moieties include nucleic acids. Shortsequences of RNA or DNA or oligonucleotides, preferably less than aboutthirty nucleotides in length and as short as four to ten nucleotides,can be detected by their ability to specifically hybridize with acomplementary nucleic acid and detected directly or indirectly using PCRwhich greatly amplifies a specific sequence that is subsequentlydetected. In a similar fashion, binding proteins and receptor-ligandcombinations are also useful as detectable labels.

[0111] The second component of the bioreactive agent is thephotoreactive moiety. The photoreactive moiety is a chemical moietycapable of forming one or more covalent bonds with a substrate which canbe cleaved with electromagnetic radiation. These bonds may be formedwith a chemical group on the substrate such as, for example, an amine,phenol, imidazole, aldehyde, carboxylic acid or thiol. The photoreactiveagent is a substituted aromatic ring containing at least one polyatomicgroup and, optionally, one or more monoatomic groups. The aromatic ringis preferably a five or six-membered ring. The substitutions comprisethe polyatomic and optional monoatomic groups. The polyatomic groupimparts electron channeling properties to attract or repel electrons tocertain locations within the chemical structure, thereby creating orestablishing the conditions to create the selectively cleavable covalentbonds. Some monoatomic groups such as halides can adjust the frequencyor wavelength of the electromagnetic radiation which will inducecleavage. As such, monoatomic groups fine tune the cleavage event tosensitize conjugates to predetermined frequencies or intensities ofradiation.

[0112] One class of photoreactive moieties are 2-nitrobenzylderivatives. In their ground state, 2-nitrobenzyl-based agents andconjugates have an intramolecular hydrogen bond between benzylichydrogen and the ortho nitro group (—CH—O₂N) (B. Brzezinski et al., J.Chem. Soc. Perkin. Trans. 2:2257-61, 1992). Upon illumination withwavelengths of greater than 300 nm, these chemical compounds transitionto an excited state. Proton transfer reaction from benzylic carbon tothe oxygen in nitro group takes place which is followed by electronrearrangement (FIG. 4). This reaction results in the formation of atransient species called an aci-nitro ion which is in a rapidequilibrium with a cyclic form. In the cyclic intermediate, electronrearrangement and oxygen transfer from nitrogen to benzylic carbon takesplace resulting in the formation of 2-nitroso derivatives and release ofa substrate which is a good leaving group (J. A. McCray et al., Annu.Rev. Biophys. Chem. 18:239-70, 1989).

[0113] Chemical synthesis of PCB NHS-ester involves three principalsteps: (1) Generation of the photoreactive moiety, for example,5-methyl-2-nitroacetophenone. (2) Generation of a suitable amino groupand attachment to biotin containing spacer. (3) Generation of hydroxylgroups and derivatization as N-hydroxysuccinimidyl carbonate(NHS-ester). These steps are schematically represented in FIG. 5.

[0114] Bioreactive agents can also be synthesized based on otherphotoreactive moieties. Chemical syntheses of two other classes ofphotocleavable moieties 3,5-dimethoxybenzyl and 2-nitrobenzenesulfenyl(FIG. 2) can be carried out using similar synthesis strategies. These2-nitrobenzyl groups all contain a benzylic carbon-hydrogen bond orthoto a nitro group, which is necessary for their photolability. In thedevelopments of these photolabile groups as protecting groups,difficulties were encountered as the subsequent reactions of thesecarbonyl compounds resulted in formation of coupled azo compounds, whichact as internal light filters (V. N. R. Pillai, Synthesis 1, 1980).These complications were overcome in the present invention with the useof α-substituted, o-nitrobenzyl compounds. Bioreactive agents of theinvention that form a detectable photocleavable conjugate can, forexample, be represented by the formula:

[0115] wherein X is selected from the group consisting of a halogen, N₂,CH₂-halogen, —N═C═O, —N═C═S, —S—S—R, NC₂H₄, —NC₄H₂O₂, —OH, —NHNH₂,—OP(OR₃)N(R₄)R₅ and —OCO—G wherein G is selected from the groupconsisting of a halogen, N₃, O-esters and N-amides; R₁, R₂, R₃, R₄ andR₅ are selected from the group consisting of hydrogen, alkyls,substituted alkyls, aryls and substituted aryls, —CF₃, —NO₂, —COOH and—COOR, and may be the same or different; A is a divalent functionalgroup selected from the group consisting of —O—, —S— and —NR₁; Ycomprises one or more polyatomic groups which may be the same ordifferent; V comprises one or more optional monoatomic groups which maybe the same or different; Q comprises an optional spacer moiety; m1 andm2 are integers from 0-5 and may be the same or different; and Dcomprises a selectively detectable moiety which is distinct from R₁-R₅.The O-ester may be cyanomethyl, o and p nitrophenyl, 2,4-dinitrophenyl,2,4,5-trichlorophenyl, pentachlorophenyl, pentafluorophenyl,N-hydroxyphthalimidyl, N-hydroxysuccinimidyl, 1-hydroxypiperidinyl,5-chloro-8-hydroxy-quinolyl, 1-hydroxybenzotriazolyl,3,4-dihydro-4-oxobenzotriazin-3-yl (DHBT),2,3-dihydro-2,5-diphenyl-3-oxo-thiophen-1,1-dioxide-4-yl (TDO),1,2-benzisoxasolyl, 2-hydroxypyridyl or derivatives or combinationsthereof. The N-amide is an imidazolyl, benzimidazolyl, benzisoxazolyl,3,5-dioxo-4-methyl-1,2,4-oxadiezolidinyl or derivatives or combinationsthereof.

[0116] Polyatomic groups can be attached to the aromatic ring includenitro groups (—NO₂), sulfoxide groups such as (—SO₃), alkyl groups suchas methyl (—CH₃) and ethyl groups (—CH₂CH₃), alkoxyl groups such as(—OCH₃), and derivatives and combinations thereof. Useful monoatomicgroups are halides such as chloride (—Cl), fluoride (−F), iodide (—I),bromide (—Br), and hydrogen (—H). These groups may be placed in any ofthe available positions around the ring. Selection and placement of thepolyatomic and monoatomic groups may influence the wavelength ofelectromagnetic radiation required to induce cleavage and the period oftime that radiation must be applied to induce efficient cleavage. Thechemical moieties at R₁, R₂, R₃, R₄ and/or R₅ may also influence thephotoreaction.

[0117] It is sometimes useful to include in the agent a spacer moietybonded between the photoreactive moiety and the detectable moiety. Thepresence of the spacer can be advantageous sterically for substratebinding. The spacer moiety (Q) may comprise a branched or straight chainhydrocarbon, a polymeric carbohydrate, or a derivative or combinationthereof. The preferred spacer moiety is represented by the formula:

[0118] wherein W and W′ are each selected from the group consisting of—C(O)—, —C(O)—NH—, —HN—C(O)—, —NH—, —O—, —S— and —CH₂—, and may be thesame or different; and n1 and n2 are integers from 0-10 which can be thesame or different and if either n1 or n2 is zero, then W and W′ areoptional. Examples of the chemical structure of bioreactive agents aredepicted in FIG. 6.

[0119] Another embodiment of the invention is directed to photocleavableconjugates comprising bioreactive agents photocleavably coupled tosubstrates. Conjugates have the property that they can be selectivelycleaved with electromagnetic radiation to release the substrate.Substrates are those chemicals, macromolecules, cells and othersubstances which are or can be used to detect and isolate targets.Substrates that are selectively cleaved from conjugates may be modifiedby photocleavage, but still functionally active, or may be released fromthe conjugate completely unmodified by photocleavage. Substrates may becoupled with agents, uncoupled and recoupled to new agents at will.

[0120] Useful substrates are any chemical, macromolecule or cell thatcan be attached to a bioreactive agent. Examples of useful substratesinclude proteins, peptides, amino adds, amino acid analogs, nucleicacids, nucleosides, nucleotides, lipids, vesicles, detergent micells,cells, virus particles, fatty acids, saccharides, polysaccharides,inorganic molecules and metals. Substrates may also comprise derivativesand combinations of these substances such as fusion proteins,protein-carbohydrate complexes and organo-metallic compounds. Substratesmay also be pharmaceutical agents such as cytokines, immune systemmodulators, agents of the hematopoietic system, recombinant proteins,chemotherapeutic agents, radio-isotopes, antigens, anti-neoplasticagents, enzymes, PCR products, receptors, hormones, vaccines, haptens,toxins, antibiotics, nascent proteins, synthetic pharmaceuticals andderivatives and combinations thereof.

[0121] Substrates may be targets or part of the targets such as an aminoacid in the synthesis of nascent polypeptide chains wherein substratesmay be amino acid or amino acid derivative which becomes incorporatedinto the growing peptide chain. Substrates may also be nucleotides ornucleotide derivatives as precursors in the synthesis of a nucleic acid.Constructs useful in creating synthetic oligonucleotide conjugates maycontain phosphoramidites or derivatives of DATP, dCTP, dTTP and dGTP,and also ATP, CTP, UTP and GTP. Resulting nucleic acid-conjugates can beused in PCR technologies, antisense therapy, and prophylactic anddiagnostic applications. Substrates may be targets, for example, when itis possible to specifically react the bioreactive agent with substratein a mixture such that the reaction creates the conjugate. Suchconjugates are useful when it is desirable to follow a target through abiological or other type of system such as when determining thehalf-life of a pharmaceutical.

[0122] Photocleavage of conjugates of the invention should preferablynot damage released substrate or impair substrate activity. Proteins,nucleic acids and other protective groups used in peptide and nucleicacid chemistry are known to be stable to most wavelengths of radiationabove 300 nm. PCB carbamates, for example, undergo photolysis uponillumination with long-wave UV light (320-400 nm), resulting in releaseof the unaltered substrate and carbon dioxide (FIG. 7). The yield andexposure time necessary for release of substrate photo-release arestrongly dependent on the structure of photoreactive moiety. In the caseof unsubstituted 2-nitrobenzyl PCB derivatives the yield of photolysisand recovery of the substrate are significantly decreased by theformation of side products which act as internal light filters and arecapable of reacting with amino groups of the substrate. In this case,illumination times vary from about 1 minute to about 24 hours,preferably less than 4 hours, more preferably less than two hours, andeven more preferably less than one hour, and yields are between about 1%to about 95% (V. N. R. Pillai, Synthesis 1, 1980). In the case ofalpha-substituted 2-nitrobenzyl derivatives (methyl, phenyl), there is aconsiderable increase in rate of photo-removal as well as yield of thereleased substrate (J. E. Baldwin et al., Tetrahedron 46:6879, 1990; J.Nargeot et al., Proc. Natl. Acad. Sci. USA 80:2395, 1983).

[0123] The choice of a particular bioreactive agent depends on whichmolecular groups of the substrate are to be derivatized. For example,reaction of photocleavable biotin NHS-ester with a protein results information of a covalent bond with primary amino groups such as at theε-position of lysine residues or the α-NH₂ group at the N-terminal of aprotein. Normally, a number of lysine residues are exposed on thesurface of a protein and available for such reaction. Alternatively,several other photocleavable biotins can be used which react withhydroxyl groups (—OH) present in tyrosine, threonine and serineresidues, carboxyl groups (—COOH) present in aspartate and glutamateresidues, and sulfhydryl groups (—SH) present in cysteine residues(Table 4). Thus, a wide variety of groups are available which are likelyto be on the surface of a target protein.

[0124] Attachment of photocleavable biotin to molecules which bindproteins such as receptor ligands, hormones, antibodies, nucleic acids,and proteins that bind glycoproteins can also be accomplished because ofthe wide variety of reactive groups available for photocleavablebiotins. For example, photocleavable biotin can be conveniently linkedto antibodies which are directed against a particular protein.Alternatively, photocleavable biotins can be linked to DNA and RNA or toa variety of small molecules including receptor ligands and hormones.The importance of biotinylation of binding-complexes for isolation ofproteins such as membrane receptors and splicesomes has already beendemonstrated using conventional biotins or non-photocleavable biotins.

[0125] The choice of the detectable moiety depends on the substrate, itsenvironment and the desired method of detection and isolation. Forexample, a substrate present in low concentrations may require asensitive method of detection such as fluorescent spectroscopy therebyrequiring a fluorescent moiety such as coumarin The wavelength offluorescent emission can be selected by the choice of detectable moietyso as not to interfere with any natural fluorophores which may bepresent in the mixture. In cases where rapid isolation of the substrateis desired, choice of the detectable moiety may be determined by theavailability of a suitable coupling agent. For example, an antigen whichserves as the detectable moiety may be used if a suitable antibody isavailable. Since the detectable moiety, the reactive group and thephotoreactive moieties are chemically separate in the bioreactive agent,the properties of each can be adjusted to meet the multiple requirementsfor detection and isolation of a particular substrate.

[0126] Conjugates of the invention may be attached to a solid supportvia the detectable moiety, the substrate or any other chemical group ofthe structure. The solid support may comprise constructs of glass,ceramic, plastic, metal or a combination of these substances. Usefulstructures and constructs include plastic structures such as microtiterplate wells or the surface of sticks, paddles, beads or microbeads,alloy and inorganic surfaces such as semiconductors, two and threedimensional hybridization and binding chips, and magnetic beads,chromatography matrix materials and combinations of these materials.Examples of the chemical structure of conjugates of the inventioninclude:

[0127] wherein SUB comprises a substrate; R₁ and R₂ are selected fromthe group consisting of hydrogen, alkyls, substituted alkyls, aryls,substituted aryls, —CF₃, —NO₂, —COOH and —COOR, and may be the same ordifferent; A is a divalent functional group selected from the groupconsisting of —O—, —S— and —NR₁; Y comprises one or more polyatomicgroups which may be the same or different; V comprises one or moreoptional monoatomic groups which may be the same or different; Qcomprises an optional spacer moiety; m1 and m2 are integers between 1-5which can be the same or different; and D comprises a selectivelydetectable moiety which is distinct from R₁ and R₂.

[0128] As discussed above, the polyatomic group may be one or more nitrogroups, alkyl groups, alkoxyl groups, or derivatives or combinationsthereof. The optional monoatomic group may be one or more fluoro,chloro, bromo or iodo groups, or hydrogen. The polyatomic and monoatomicgroups and the chemical moieties at R₁ and R₂ may effect thephotocleavage reaction such as the frequency of radiation that willinitiate photocleavage or the the exposure time needed to execute acleavage event. The spacer moiety (Q) may be a branched or unbranchedhydrocarbon or a polymeric carbohydrate and is preferably represented bythe formula:

[0129] wherein W and W′ are each selected from the group consisting of—CO—, —CO—NH—, —HN—CO—, —NH—, —O—, —S— and —CH₂—, and may be the same ordifferent; and n1 and n2 are integers from 0-10 which can be the same ordifferent and if either n1 or n2 is zero, then W and W′ are optional.Specific examples of conjugates of the invention are depicted in FIG. 8.

[0130] Another embodiment of the invention is directed to conjugateswhich are pharmaceutical compositions. Compositions must be safe andnontoxic and can be administered to patients such as humans and othermammals. Composition may be mixed with a pharmaceutically acceptablecarrier such as water, oils, lipids, saccharides, polysaccharides,glycerols, collagens and combinations thereof and administered topatients.

[0131] Pharmaceutical compositions with photo-releasable substrates areuseful, for example, for delivery of pharmaceutical agents which haveshort half-lives. Such agents cannot be administered through currentmeans without being subject to inactivation before having an effect.Pharmaceutical agents in the form of conjugates, covalently bound tobioreactive agents, are more stable than isolated agents. After generaladministration of the composition to the patient, the site to be treatedis exposed to appropriate radiation releasing substrate which producesan immediate positive response in a patient. Uncoupling from thebioreactive agent at the point of maximal biological effect is anadvantage unavailable using current administration or stabilizationprocedures. In an analogous fashion, other areas of the patient's bodymay be protected from the biological effect of the pharmaceutical agent.Consequently, using these conjugates, site-directed and site-specificdelivery of a pharmaceutical agent is possible.

[0132] Another embodiment of the invention is directed to a method forisolating targets from a heterologous mixture. Bioreactive agents arecontacted with the mixture to react with target forming the conjugate.Alternatively, conjugates can be contacted with the heterologous mixtureto couple substrate within the conjugate to one or more targets.Conjugates can be separated from the mixture by any currently availabletechniques (e.g. Table 5). TABLE 5 Affinity Techniques Using AvidinMaterial Method of Separation Magnetic beads coated with Magneticseparation Streptavidin Beads coated with Streptavidin Washing (e.g.centrifugation) and elution Biotinylated Antibodies ImmunoprecipitationCross-linked-bisacrylamide/azolactone Column Chromatography copolymerswith avidin Agarose coated with Streptavidin Column Chromatography

[0133] Procedures such as chemical or physical separation of componentsof the mixture, electrophoresis, electroelution, sedimentation,centrifugation, filtration, magnetic separation, chemical extraction,affinity separation methods such as affinity chromatography or anotherchromatographic procedure such as ion-exchange, gradient separation,HPLC or FPLC, and combinations of these techniques are well-known andallow for a rapid isolation with a high efficiency of recovery (e.g. M.Wilchek et al., Methods Enzymol. 184, 1990; M. Wilchek et al., Anal.Biochem 171:1, 1988). After separation or isolation, targets can beeasily quantitated using available methods such as optical absorbance ortransmission (e.g. nucleic acid, proteins, lipids) or the Bradford (M.Bradford, Anal. Biochem. 72:248, 1976) or Lowry (O. Lowry et al., J.Biol. Chem. 193:265, 1951) assays (e.g. proteins), both of which arecommercially available. After separation, coupled conjugates are treatedwith electromagnetic radiation to release substrate. The substratetargets can than be separated from the released bioreactive agent, ifdesired, to obtain substantially or completely pure targets.

[0134] Targets which can be detected and isolated in a highly purifiedform by this method include nearly any chemical, molecule ormacromolecule including immune system modulators, agents of thehematopoietic system, cytokines, proteins, hormones, gene products,antigens, cells including fetal and stem cells, toxins, bacteria,membrane vesicles, virus particles, and combinations thereof. Detectionand isolation are determined by the ability of the bioreactive agent tobind substrate. For example, nucleic acids can be base-paired tocomplementary nucleic acids, to nucleic acid binding proteins or tochemical moieties which react specifically with chemical moieties foundon nucleic acids. Proteins can be bound with monoclonal or polyclonalantibodies or antibody fragments specific to those proteins, or chemicalmoieties which react specifically with chemical moieties found on theproteins of interest. Substrates may be, for example, precursors oftargets such as one or more of the naturally or non-naturally occurringamino acids wherein the target is a nascent protein, or one or moreribonucleotides, deoxyribonucleotide or primers when the target is anucleic acid. Precursor can be incorporated into target molecules by,for example, in vivo or in vitro replication, transcription ortranslation. Target may be a protein or protein-containing complex,nucleic acid, gene sequence or PCR product. Substrates may also bereceptors which bind to or otherwise associate with ligands specific forthe receptor molecules. Receptors which can be isolated includecytokines wherein the target is a cytokine receptor and antigens whereinthe target is an antibody. Preferred conjugates for the detection andisolation of a target from a heterologous mixture are photocleavablebiotins linked to antibodies (polyclonal, monoclonal, fragments),photocleavable coumarins linked to antibodies, photocleavable dansylslinked to lipids and derivatives and modifications thereof.

[0135] The heterologous mixture which contains target may be abiological sample, any proteinaceous composition such as a cellular orcell-free extract, nucleic acid containing compositions, a biomasscontaining, for example, vegetative or microbial material, a cellculture of primary or immortalized cells, lipid vesicles or evenanimals. Animals may be used to detect targets which may be present inthe body or parts of the body or, alternatively, to collect and isolatetargets such as macromolecules or cells from animal models. Substratecan also be proteins, peptides, amino acids, amino acid analogs,nucleosides, nucleotides, lipids, vesicles, detergent micells, fattyacids, saccharides, polysaccharides, inorganic molecules, metals andderivatives and combinations thereof

[0136] In an application of this method, the substrate may be anintegral component of the target such as a nucleotide in the detectionand isolation of nascent nucleic acids or an amino acid in the detectionand isolation of nascent proteins. Substrate is incorporated into targetby chemical or enzymatic techniques and detected and isolated by thepresence of the detectable moiety. Briefly, conjugates are contactedwith reagents in a heterologous mixture such as, for example, in areplication, transcription, translation or coupledtranscription/translation system. Substrates are incorporated intotargets through the action of components in the system such as enzymes,precursor molecules and other reagents of the system. Conjugate coupledtargets are separated from the mixture and treated with electromagneticradiation to release the .target which is then isolated.

[0137] Conjugates can be contacted with a heterologous mixture byincubation as in, for example, the enzymatic incorporation of amacromolecular precursor into a nascent macromolecule which may beeither in vivo or in vitro. Nucleic acid polymerases will incorporateprecursor nucleotides or nucleic acid primers into nucleic acids. Invitro incubations in cell-free reaction mixture are typically performedat a temperature of between about 4° C. to about 45° C., preferably atbetween about 12° C. to about 37° C., and more preferably at about roomtemperature. Incubation of conjugates into nascent macromolecules may becomplete in about 5 minutes, about 15 minutes, or about one hourdepending on the incubation conditions, or may require two, three ormore hours to complete. When the heterologous mixture is an animal or ananimal model in vivo incubations are generally performed at bodytemperature and may require hours or days for conjugates to distributeto areas of the animal's body which may be remote from the site ofintroduction, for conjugates to react with targets and for conjugatescoupled with targets to be collected.

[0138] One of the preferred embodiments of the invention relates to thedetection or isolation of protein using photocleavable biotin. In oneapplication of this embodiment, PCB is reacted with a protein throughthe formation of covalent bonds with specific chemicals groups of theprotein forming a conjugate. The protein may be either the target to beisolated or detected or a probe for the target protein such as anantibody. The target protein can then be isolated using streptavidinaffinity methodology.

[0139] Another application of this embodiment is directed to the use ofphotocleavable biotin to isolate nascent proteins that can be createdfrom in vitro or in vivo protein synthesis. Basically, photocleavablebiotins are synthesized and linked to amino acids (PCB-amino acids)containing special blocking groups. These conjugates are charged to tRNAmolecules and incorporated into peptides and proteins using atranslation or coupled transcription/translation system. PCB-amino acidsof the invention have the property that once illuminated with light, aphotocleavage occurs that produces a native amino acid plus the freebiotin derivative. Such proteins can be isolated in a structurallyand/or functionally unaltered form.

[0140] The detailed procedure for the production of photocleavablebiotin amino acids and their incorporation into the nascent proteinsinvolves a few basic steps. First, photocleavable biotin is synthesizedand linked to an amino acid with an appropriate blocking group. ThesePCB-amino acid conjugates are charged to tRNA molecules and subsequentlyincorporated into nascent proteins in an in vivo or in vitro translationsystem. Alternatively, a tRNA molecule is first charged enzymaticallywith an amino acid such as lysine which is then coupled to a reactivePCB. Nascent proteins are separated and isolated from the othercomponents of synthesis using immobilized streptavidin. Photocleavage ofPCB-streptavidin complex from the nascent protein generates a pure andnative, nascent protein.

[0141] PCB is attached to an amino acid using, for example, theside-chain groups such as an amino group (lysine), aliphatic andphenolic hydroxyl groups (serine, threonine and tyrosine), sulfhydrylgroup (cysteines) and carboxylate group (aspartic and glutamic acids)(FIG. 9). Synthesis can be achieved by direct condensations withappropriately protected parent amino acids. For example, lysine sidechain amino group can be modified with PCB by modification of theε-amino group. The synthesis of, for example, PCB-methionine involvesprimarily α-amino group modification. PCB-methionine can be charged toan initiator tRNA which can participate in protein synthesis only atinitiation sites which results in single PCB incorporation per copy ofthe nascent protein.

[0142] One method for incorporation of a photocleavable biotin aminoacid into a nascent protein involves misaminoacylation of tRNA Normally,a species of tRNA is charged by a single, cognate native amino acid.This selective charging, termed here enzymatic aminoacylation, isaccomplished by enzymes called aminoacyl-tRNA synthetases and requiresthat the amino acid to be charged to a tRNA molecule be structurallysimilar to a native amino acid. Chemical misaminoacylation can be usedto charge a tRNA with a non-native amino acids such as photocleavableamino acids. The specific steps in chemical misaminoacylation of tRNAsare depicted in FIG. 10.

[0143] As shown, tRNA molecules are first truncated to remove the3′-terminal residues by successive treatments with periodate, lysine (pH8.0) and alkaline phosphate (Neu et al., J. Biol. Chem. 239:2927-34,1964). Alternatively, truncation can be performed by geneticmanipulation, whereby a truncated gene coding for the tRNA molecule isconstructed and transcribed to produce truncated tRNA molecules (Sampsonet al., Proc. Natl. Acad. Sci. USA 85:1033, 1988). Second, protectedacylated dinucleotides, pdCpA, are synthesized (Hudson, J. Org. Chem.53:617, 1988; E. Happ, J. Org. Chem. 52:5387, 1987). PCB-amino acidsblocked appropriately at their side chains and/or at α-amino groups,using standard protecting groups like Fmoc, are prepared and coupledwith the synthetic dinucleotide in the presence of carboxy groupactivating reagents. Subsequent deprotection of Fmoc groups yieldsaminoacylated dinucleotide.

[0144] Third, the photocleavable biotin amino acid is ligated to thetruncated tRNA through the deprotected dinucleotide. The bond formed bythis process is different from that resulting from tRNA activation by anaminoacyl-tRNA synthetase, however, the ultimate product is the same. T4RNA ligase does not recognize the O-acyl substituent, and is thusinsensitive to the nature of the attached amino acid (FIG. 10).Misaminoacylation of a variety of non-native amino acids can be easilyperformed. The process is highly sensitive and specific for thestructures of the tRNA and the amino acid.

[0145] Aminoacylated tRNA linked to a photocleavable biotin amino acidcan also be created by employing a conventional aminoacyl synthetase toaminoacylate a tRNA with a native amino acid or by employing specializedchemical reactions which specifically modify the native amino acidlinked to the tRNA to produce a photocleavable biotin aminoacyl-tRNAderivative. These reactions are referred to as post-aminoacylationmodifications. Such post aminoacylation modifications do not fall underthe method of misaminoacylation, since the tRNA is first aminoacylatedwith its cognate described amino acid.

[0146] In contrast to chemical aminoacylation, the use ofpost-aminoacylation modifications to incorporate photocleavable biotinnon-native amino acids into nascent proteins is very useful since itavoids many of the steps including in misaminoacylation. Furthermore,many of the photocleavable biotin derivatives can be prepared which havereactive groups reacting specifically with desired side chain of aminoacids. For example, postaminoacylation modification oflysine-tRNA^(Lys), an N-hydroxysuccinimide derivative of PCB canprepared that would react with easily accessible primary ε-amino andminimize reactions occurring with other nucleophilic groups on the tRNAor α-amino groups of the amino acylated native amino acid. These othernon-specific modifications can alter the structure of the tRNA structureand severely compromise its participation in protein synthesis.Incomplete chain formation could also occur when the α-amino group ofthe amino acid is modified. Post-aminoacylation modifications toincorporate lysine-biotin non-native amino acids into nascent proteinshas been demonstrated (tRNA^(nacend)™; Promega; Madison, Wis.) used forthe detection of nascent protein containing biotin using Western Blotsfollowed by enzymatic assays for biotin (T. V. Kurzchalia et al., Eur.J. Biochem. 172:663-68, 1988). However, these biotin derivatives are notphotocleavable which, in the case of NHS-derivatives of PCB, allows thebiotin linkage to the lysine to be photochemically cleaved.

[0147] PCB-amino acids can also be incorporated into polypeptide bymeans of solid-support peptide synthesis. First, PCB-amino acids arederivatized using base labile fluorenylmethyloxy carbonyl (Fmoc) groupfor the protection of α-amino function and acid labile t-butylderivatives for protection of reactive side chains. Synthesis is carriedout on a polyamide-type resin. Amino acids are activated for coupling assymmetrical anhydrides or pentafluorophenyl esters (E. Atherton et al.,Solid Phase Peptide Synthesis, IRL Press, Oxford, 1989). Second, aminoacids and PCB are coupled and the PCB-amino acid integrated into thepolypeptide chain. Side chain PCB-derivatives, like ε-amino-Lys, sidechain PCB-amino acid esters of Glu and Asp, esters of Ser, Thr and Tyr,are used for incorporation at any site of the polypeptide. PCB-aminoacids may also be incorporated in a site-specific manner into the chainat either predetermined positions or at the N-terminus of the chainusing, for example, PCB-derivatized methionine attached to the initiatortRNA.

[0148] A wide range of polypeptides can be formed from PCB-amino acidscytokines and recombinant proteins both eukaryotic and prokaryotic (e.g.α-, β- or γ-interferons; interleukin-1, -2, -3, etc.; epidermal,fibroblastic, stem cell and other types of growth factors), and hormonessuch as the adrenocorticotropic hormones (ACTHs), insulin, theparathyroid hormone (bPTH), the transforming growth factor β (TGF-β) andthe gonadotropin releasing hormone (GnRH) (M. Wilchek et al., MethodsEnzymol. 184:243, 1990; F. M. Finn et al., Methods Enzymol. 184:244,1990; W. Newman et al., Methods Enzymol. 184:275, 1990; E. Hazum,Methods Enzymol. 184:285, 1990). These hormones retain their bindingspecificity for the hormone receptor. One example is the GnRH hormonewhere a biotin was attached to the epsilon amino group Lys-6 throughreaction of a d-biotin p-nitophenyl ester. This biotinylated hormone canbe used for isolation of the GnRH receptor using avidin coated columns.

[0149] After incorporation or attachment of PCB into a protein,protein-complex or other amino acid-containing target, the target isisolated using a simple four step procedure (FIG. 11). First, abioreactive agent (PCB) is synthesized. Second, a substrate is coupledto the bioreactive agent forming a conjugate. Third, target is separatedfrom other materials in the mixture through the selective interaction ofthe photocleavable biotin with avidin, streptavidin or theirderivatives. Captured targets may be immobilized on a solid support suchas magnetic beads, affinity column packing materials or filters whichfacilitates removal of contaminants. Finally, the photocleavable biotinis detached from the target by illumination of a wavelength which causesthe photocleavable biotin covalent linkage to be broken. Targets aredissolved or suspended in solution at a desired concentration. In thosesituations wherein conjugate coupled targets are not attached to solidsupports, release of targets can be followed by another magnetic captureto remove magnetic particles now containing avidin/streptavidin boundbiotin moiety released form the photocleavage of PCB. Thus, a completelyunaltered protein is released in any solution chosen, in a purified formand at nearly any concentration desired.

[0150] Another example for the use of PCB is where the conjugatecomprises a PCB-coupled antibody. The use of photocleavable biotinprovides a means for recovering target molecule and the antibody forsubsequent use. Release of a protein from binding complex can beperformed subsequent to release of the binding complex from theimmobilized streptavidin. This is an advantage since it enables therelease to be performed under well controlled conditions. For example,elution of a target protein from an affinity column often requireschanges in buffer and/or use of a competitive agent such as an epitopewhich competes for an antibody binding site. This can require longexposure of the protein to damaging conditions or the need for increasedamounts of the competitive agent which can be prohibitively expensive.In contrast, once the protein complex is removed from the immobilizedstreptavidin by photocleavage, the complex can be separated moreconveniently. In the case where antibodies are used as the substrate, anadditional advantage of the invention is that the antibody can also berecovered in an unaltered and purified form.

[0151] A simple scheme for using a PCB-antibody is shown in FIGS. 12Aand 12B. Target protein/antibody conjugated to photocleavable biotin isthen immobilized with streptavidin. Target protein/antibody is decoupledby illumination with light and the target protein/antibody released.Target protein is separated from antibody using, for example, increasedor decreased salt (NaCl, KCl) concentrations and recovered.Alternatively, as shown in FIG. 12B, the target is removed from theantibody by using an epitope-PCB conjugate which competes for theantibody binding site. The antibody-epitope-PCB conjugate is thenisolated from the target by immobilization with streptavidin. Theantibody-epitope complex is then released by photocleaving thebiotin-epitope complex. A wide variety of other molecules that interactwith proteins can also be utilized in conjunction with PCB for proteinisolation, as shown in Table 2, including polypeptides, proteincomplexes and small ligands.

[0152] PCB derivatives attached or incorporated into antibodies or othermacromolecules such as DNA which serve as hybridization probes can alsobe used advantageously for sequential multiple detection of targets. Asin the case of conventional assays for target based onstreptavidin-biotin interactions, the presence of the target is signaledby a streptavidin-enzyme complex which binds to the biotinylated probeand produces an amplified signal by converting a substrate into aproduct which is easily detectable due to a distinctive physicalproperty such as color or luminescence. However, in contrast toconventional biotins, the PCB derivatives can be completely removedallowing for separation removal of the streptavidin-enzyme complex andsubsequent addition of new probes and streptavidin complexes for thedetection of additional target molecules. A similar advantage exists forcytochemical labeling based on streptavidin-biotin interaction. In thiscase, the label can be completely removed by light, allowing foradditional specific cytochemical labels to be used.

[0153] In another application of the preferred embodiment,photocleavable biotin or a PCB derivative can be incorporated into a DNA(deoxyribonucleic acid), RNA (ribonucleic acid) or PNA (polynucleicamide; P. E. Nielsen et al., Sci. 254:1497-1500, 1991) molecule producedby, for example, chemical synthesis, PCR, nick translation or DNA or RNApolymerases (Table 6). Target DNA or RNA can then be isolated by usingstreptavidin affinity methods similar to methods discussed above.Isolation is accomplished, for example, using commercially availablemagnetic beads coated with streptavidin. Beads will bind tightly to allDNA and RNA containing the PCB derivative, whereas all other moleculesare washed away. The photocleavable biotin is then removed from thetarget nucleic acid by illumination.

[0154] The incorporation of PCB into nucleic acids such as DNA and RNAinvolves the synthesis and use of compounds that are formed from thederivatization of nucleotides with photocleavable biotins. Examples ofthe nucleotides modified using photocleavable biotin are shown FIG. 13A.

[0155] Non-specific incorporation of PCB moieties into nucleic acids isachieved, for example, using bisulfite catalyzed cytosine transaminationand subsequent reaction with PCB-NHS ester or PCB-NH-NH₂(PCB-hydrazide).Alternatively, 5′-phosphate can be converted into phosphoramidite byreaction with water soluble carbodiimide, imidazole and diamine and theresulting phosphoramidite reacted with PCB-NHS ester.

[0156] Numerous methods have been developed to introduce an amino groupon 5′-end of protected oligonucleotides during solid support synthesis.These include carbonyl diimidazole mediated modification of 5′-OH withdiamine and introduction of aliphatic amine moiety on 5′- or 3′-endduring synthesis using special phosphoramidites. Bifunctionalphosphoramidites have also been developed that allow for incorporationof reactive amino group into multiple sites in syntheticoligonucleotides. These methods produce oligonucleotide products bearingaliphatic amino groups, which can be easily converted intoPCB-carbamates by reaction with respective PCB-NHS esters. In ananalogous manner as conventional biotin, PCB moieties can be directlyincorporated into oligonucleotides during solid support synthesis usingrespective PCB-phosphoramidites.

[0157] Synthetic oligonucleotides of predetermined or random sequenceshave a variety of uses as, for example, primers, hybridization probesand antisense sequences. The synthesis of DNA and RNA oligonucleotidesutilizes phosphoramidite chemistry and is routinely performed on anautomated synthesizer with the growing nucleic acid chains attached to asolid support such as CPG (controlled pore glass). In this manner,phosphoramidites and other reagents can be added in excess and removedby filtration The synthesis cycle comprises four reactions. First, acidlabile trityl groups are removed from the 5′-OH groups. Second,phosphoramidites are coupled to the 5′-OH. Third, unreacted 5′-OH groupsare protected by capping with acetyl groups. Finally, internucleotidelinkage is converted from phosphite to phosphotriester by oxidation.This cycle is repeated until the desired sequence is obtained afterwhich, oligonucleotide is cleaved from solid support and purified using,for example, gel electrophoresis and HPLC.

[0158] Coupling efficiency for each step is preferably as high aspossible such as greater than 90% or about 97-99%. Unreacted moleculesare eliminated at each step by capping with acetyl groups preventing theformation of undesired sequences. Crude oligonucleotide contains besidesfull length sequence numerous shorter sequences also called the failuresequences. Purification of such a complex mixture is difficultespecially when it comes to isolation of full-length sequence fromslightly shorter failure sequences (e.g. n−1; n−2). This problem becomeseven more difficult when synthesizing long sequences of DNA or RNA wherecoupling efficiencies are lower and the number of failure sequenceshigher. 5′-PCB-phosphoramidites can be used to selectively labilefull-length oligonucleotides at their 5′-end during solid-supportsynthesis on automated nucleic acid synthesizers.

[0159] PCB-phosphoramidites can contain the PCB moiety linked to aphosphoramidite functionality through a spacer arm allowing forefficient binding to avidin. This reagent selectively reacts with the5′-OH group on a sugar ring and can be used on automated synthesizers.Biotinylated sequences can also be prepared using standard synthesiscycles with coupling efficiency being monitored by trityl analysis.Typical coupling efficiencies are between about 90-95%. In addition,photocleavage of 5′-PCB nucleic acid results in formation of5′-phosphorylated sequences. 5′-phosphorylated oligonucleotides arerequired for most applications in molecular biology.

[0160] Alternatively, PCB moieties can also be incorporated duringsynthesis into oligonucleotides at any position including the3′-terminus, and into multiple sites using PCB-phosphoramiditesutilizing, for example, bifunctional non-nucleosidic backbones. Enzymesincluding the Taq DNA polymerase used in PCR reactions, and other DNAand RNA polymerases are capable of incorporating biotinylatednucleotides into nucleic. acids. PCR technology comprises the process ofamplifying one or more specific nucleic acid sequences in a nucleic acidusing primers and agents for enzymatic polymerization followed bydetection of the now amplified sequence (R. K. Saild et al., Sci.230:1350, 1985; T. J. White et al., Trends Gent. 5:185, 1989). The basictechniques are described in U.S. Pat. No. 4,683,195, which isspecifically incorporated by reference, and variations thereof describedin U.S. Pat. Nos. 5,043,272, 5,057,410 and 5,106,727, which are alsospecifically incorporated by reference. Several examples exist wherebiotinylated nucleotides have been efficiently incorporated during thePCR applications. These studies demonstrate that PCR carried out in thepresence of PCB-nucleotides results in a large amplification of targetDNA fragment and simultaneous labeling with PCB. The primers requiredfor PCR reaction may also be labeled.

[0161] Table 6 lists different enzymatic methods that would allowincorporation of PCB-nucleotides to generate labeled probes for manyapplications like in situ hybridization and PCR. TABLE 6 EnzymaticMethods for Incorporation of PCB-Nucleotides Method Substrate EnzymeRemarks Nick Translation DNA or E. coli DNA pol I Most popular RNAmethod Replacement double T4 DNA High specific synthesis using T4stranded Polymerase incorporation in DNA polymerase DNA dsDNA ReverseRNA Molony murine Preparation of transcriptase leukemia virus long cDNAcopies reverse of RNA transcriptase 3′-Terminal ds DNA terminal3′-hydroxyl labeling deoxynucleotidyl terminal labeling transferase ofdsDNA RNA labeling RNA SP6 Polymerase T3 RNA is the T3 or T7 RNA mostefficient for Polymerase RNA labeling Post-transcription RNA SP6, T3 orT7 Pol Incorporate labeling allylamine-UTP first followed by itsmodification by PCB 3′-labeling of RNA RNA (in- T4 RNA ligase Based onADP cluding derivatives of PCB. tRNAs etc)

[0162] Biotin-avidin technology is currently used extensively in thefield of molecular biology and biomedicine as a means for efficientlyisolating the products of DNA and RNA synthesis as well as for detectionof specific sequences in nucleic acids. Isolation normally involves theattachment or incorporation of biotin into the DNA or RNA followed byseparation through the interaction of the biotin with streptavidin. Forexample, this methodology is used widely for the isolation of DNA, whichis the product of the polymerase chain reaction. Detection typicallyinvolves the preparation of biotin labeled nucleic acid probes. Theseprobes have found wide-spread application in gene structure and genefunction studies, the diagnosis of human, animal and plant pathogens,and the detection of human genetic abnormalities.

[0163] However, conventional methodologies are limited by the difficultyof detaching or releasing biotin from the nucleic acid. In particular,it is highly desirable to obtain unaltered DNA or RNA after it isseparated by the biotin-avidin interaction. For example, the presence ofbiotin on the nascent DNA can interfere with its subsequent utilizationin cloning or hybridization analysis. In addition, the inability toremove biotin from a biotinylated nucleic acid probe after an enzymelinked assay prevents additional hybridization assays from beingperformed on the same sample.

[0164] The utilization of photocleavable biotins in the isolation ordetection of nucleic acids eliminates many of the difficulties listedabove by providing a rapid and effective method of removing the biotinin a single step. In the case of biotin-avidin based isolation of DNAthis also accomplishes the step of releasing the DNA from theimmobilized avidin.

[0165] In a preferred embodiment of this invention, the isolation ofnucleic acids is based on three basic steps. First, a photocleavablebiotin derivative is attached to a nucleic acid molecule by enzymatic orchemical means or, alternatively, by incorporation of a photocleavablebiotin nucleotide into a nucleic acid by enzymatic or chemical means.The choice of a particular photocleavable biotin depends on whichmolecular groups are to be derivatized on the nucleic acid. For example,attachment of photocleavable biotin to a nucleic acid can beaccomplished by forming a covalent bond with the aromatic amine, sugarhydroxyls or phosphate groups (Table 4). PCB can also be incorporatedinto oligonucleotides through chemical or enzymatic means. Next, thenucleic acid molecule is separated through the selective interaction ofthe photocleavable biotin with avidin, streptavidin or their derivativeswhich can be immobilized on a material such as magnetic beads, affinitycolumn packing materials or filters. Methods for the separation ofnucleic acids from other molecules in a complex mixture usingphotocleavable biotin are well-established and similar to the moreconventional methods utilizing non-cleavable biotin. This typicallyinvolves an affinity technique based on streptavidin-biotin interactionwhereby the nucleic acid containing biotin is immobilized due to itsinteraction with streptavidin. These techniques include, as shown inTable 5, streptavidin-coated magnetic beads, streptavidin-sepharosecolumns and streptavidin conjugated filters, all of which arecommercially available. For example, nucleic acid molecules containingPCB either through attachment or incorporation are isolated usingstreptavidin-coated magnetic beads. The pool of unbound biomolecules isthen: washed to remove other reactants, buffer and salts. Finally, thephotocleavable biotin is detached from the nucleic acid by illuminationat a wavelength which causes the photocleavable biotin covalent linkageto be broken. The bioreactive agent can be removed leaving asubstantially or completely pure nucleic acid.

[0166] Another aspect of the invention is directed to the use ofphotocleavable conjugates in conjunction with PCR amplification. Methodsfor the isolation of a PCR product may use one or more oligonucleotideprimers as substrates. The nucleic acid sequence of the target is PCRamplified using the conjugated primers. Covalent bonds between theprimer and the bioreactive agent are selectively cleavable withelectromagnetic radiation to release the amplified sequences. Nucleotidesequences which can be selectively amplified by this method includesnucleotide sequences found in biological samples, bacterial DNA andeukaryotic DNA. In contrast to conventional biotins, PCB offers aneffective method to completely remove biotinylated DNA product of thepolymerase chain reaction and provides a means to simultaneously releasethe PCR product from the avidin or streptavidin binding medium andremove the biotinylation in a single step.

[0167] Other advantages over conventional biotins include theelimination of the need for special reagents or buffers. Afterphotocleavage of biotin from the PCR product, the resulting DNA issuitable for cloning and other common usages in molecular biology. Afterphotocleavage of biotin from the PCR product, it can be accuratelyanalyzed with standard analytical methods such as gel electrophoresis.Hybridization probes containing PCB can be sterilized, wherein thebiotin is completely removed so that the target DNA can be reprobedusing a second biotinylated probe. The PCB incorporated into DNA retainsthe high binding affinity to avidin unlike the several derivatives ofbiotin where properties of biotin are attenuated for the purposes ofeasy release (e.g. iminobiotin).

[0168] Two methods for the isolation of PCR products using PCB arerepresented in FIGS. 14A and 14B. In both methods, the source of theinitial pool of cells from which the target DNA is to be amplified canbe from a wide variety of sources including peripheral blood and biopsytissues. After cell lysis, the crude extract of total genome issubjected to the PCR.

[0169] In method A (FIG. 14A), DNA primers are synthesized from theflanking sequences of the target DNA with photocleavable biotinincorporation at the 5′ ends. For this purpose, a PCB-phosphoramiditecan be introduced directly at the 5′ end of the oligonucleotide primerduring DNA synthesis. A set of PCR cycles is carried out using normaldNTPs. This procedure results in PCR product where the photocleavablebiotin is present on the 5′ end of each complementary strand. PCRproducts are separated from the mixture containing other components ofthe PCR mixture including nucleotides, enzymes, buffers by usingstreptavidin such as present on coated magnetic beads (e.g. DynabeadsM-280 Streptavidin) which bind the photocleavable biotin present at the5′ end of the DNA. A typical procedure to be followed is to mix 40 μl ofwashed Dynabeads M-280 Streptavidin with 40 μl of the PCR mixture and toincubate for 15 minutes at room temperature. The Dynabeads are thencollected using magnetic means such as the DYNAL Magnetic ParticleConcentrators. Residual primers will be bound to thestreptavidin-binding material due to the presence of photocleavablebiotin at its 5′ end. A small spin column like NAP-5 (Pharmacia Biotech;Piscataway, N.J.) can be used to remove smaller molecules and primersbefore streptavidin-magnetic bead capture is carried out. Theimmobilized PCR product, now bound to streptavidin, is photolyzed torelease biotin from the DNA and the unmodified PCR product recovered.

[0170] In method B (FIG. 14B), the problem of primers contamination iseliminated. DNA primers are synthesized for the flanking sequences ofthe target DNA without 5′ biotinylation using conventionaloligonucleotide synthesis. Normal dNtPs along with a small pool ofPCB-dUTP are introduced into the PCR reactions. For convenience, thedNTPs and PCB-dUTP can be premixed into aliquots to be used inconjunction with PCR reactions. The PCR products are separated from themixture containing other components of the PCR mixture includingnucleotides, enzymes, buffers and primers by using streptavidin such aspresent on coated magnetic beads (e.g. Dynabeads M-280 Streptavidin).

[0171] The immobilized PCR product, now bound to streptavidin, isphotolyzed to release biotin from the DNA and recover unmodified PCRproduct. Method A may sometimes be preferable when the immobilized DNAwhich is bound to streptavidin is to be assayed using a biotinylatedprobe. In this case the entire complex could be released after assay byphotocleavage followed by an addition separation step, eliminating thebiotinylated probe and leaving the PCR product free for further use suchas for cloning. Alternatively, Method B may be preferable if release ofa primer-free product is required without an intermediate assay. Thiswould also produce a higher recovery since there are more PCB moleculesper molecule of DNA.

[0172] PCR is also widely used in the detection of a variety of diseasesrelated markers. Table 7 illustrates the various uses of PCR fordetection of diseases and disorders and the potential uses ofPCB-incorporated PCR in such diagnostic and forensic applications. TABLE7 PCR use for detection of disease related DNA/RNA Assay of the PCRDisease/Virus/Bacteria Primer product HTLV-I various targets in- Liquidhybridization/Spot cluding tax, gag or env blot HIV targeted at the con-Oligomer Hybridization served regions of the virus Hepatitis-B pol geneSouthern hybridization Papillomaviruses Dot-blot Restriction enzymeanalysis Cytomegalovirus Hybridization (herpes virus group) Enterovirus100% conserved RT-PCR followed by regions RNA hybridizationGastroenteritis Reverse hybridization Cholera

[0173] In a preferred embodiment of the invention, the methodology,based on PCB, for isolation and detection of PCR products can be appliedto diagnostic assays of a variety of diseases, the detection ofmutations as well as to the identification of unique DNA sequences. Forexample, serological assays such as Western blots and immunofluorescenceand radioimmunoprecipitation assays provide a rapid and sensitiveprocedure to screen for the presence of antibodies to HIV-1. Further, incurrent PCR-based analysis, highly conserved regions of the viralgenomes are targeted for amplification and involve hybridization using³²P-labeled oligomer probes in solution to one strand of an amplifiedproduct. These tests can be used only for the direct detection of thevirus. A useful assay for the detection of HIV would detect not onlyactive virus, but also the presence of latent virus which has not yetexpressed its genome, but is still present in cells. This would allowdetermination of both latent and actively replicating virus. This wouldbe particularly useful in newborns where maternal antibodies caninterfere with serological tests.

[0174] In another preferred embodiment, conjugates may be used toefficiently create genomic or cDNA libraries. Construction of aPCR-directed cDNA library from total RNA may provide the onlymethodological approach to analyze cell-specific gene expression wherethe amount of biological tissue is severely restricted. This approach isparticularly applicable where a specific stimulus results in themodification or differentiation of a small number of cells within apopulation.

[0175] Current schemes for the construction of, for example, cDNAlibraries require the isolation of cellular RNA and usually furtherpurification of mRNA from the more abundant rRNA and tRNA components.The major advantage in PCR based methods is that mRNA purification isnot necessary. This is particularly advantageous where biologicalmaterial is limited wherein efficient mRNA purification would beimpossible. The general strategy is illustrated schematically in FIG.15.

[0176] High quality, intact RNA is prepared using guanidiniumhydrochloride procedure (S. J. Gurr et al., PCR: A Practical Approach,Oxford University Press, New York, 1991). First strand cDNA issynthesized using AMV reverse transcriptase in the presence of PCB-dCTPin 1:1 ratio with dCIP (final concentration of both should equal that ofother dNTPs). This is followed by removal of oligo-dT primers which canbe carried out in a rapid and quantitative manner using magnetic capturefollowed by illumination. Current methods use CTAB precipitation whichresults in loss of valuable cDNA:RNA hybrids, which is followed byhomopolymer tailing using oligo-dG (A. Otsuka, Gene 13:339, 1981). Afterhomopolymer tailing, the RNA is hydrolyzed which subjects cDNA to harshcondition such as 50 mM NaOH at 65° C. These steps are not necessaryusing PCB-nucleotide conjugates. Second strand synthesis and cDNAamplification is achieved by PCR in presence of PCB-nucleotides. All thePCR products are ethanol precipitated and are ligated with a suitablydigested vector. These vectors are rapidly purified by using magneticcapture and illumination. These vectors can be screened usingPCB-modified hybridization probes to selectively remove the vector ofinterest. This vector can then be magnetically captured and illuminatedto obtain pure vector containing the DNA of interest.

[0177] Another aspect of the invention facilitates the process ofsite-directed mutagenesis. For example, cassette mutagenesis is apowerful approach in creating site directed mutants and avoidssequencing of entire genes to confirm the introduction of a mutation.Basic steps in cassette mutagenesis require construction of a vectorwhich contains the gene of interest with well-separated, uniquerestriction sites. Restriction digestion of vector carrying the gene ofinterest using two unique restriction enzymes to remove a cassette ofdouble-stranded DNA where the mutation is to be introduced.

[0178] The cassette containing the desired mutation is synthesized usingautomated oligonucleotide synthesis. The digested vector and thecassette are ligated to generate complete vector. These ligated mixturesare transformed into host cells and the colonies are screened bysequencing the cassette regions of plasmid mini-preps. Although thisprocess is capable of rapidly and accurately generating a large numberof site directed mutants as shown in case of bacteriorhodopsin (H. G.Khorana, J. Biol. Chem. 263:7439, 1988), there are several areas wheretime and resources can be saved using PCB.

[0179] After the initial vector restriction, the new mutant containingcassette is labeled either chemically or enzymatically with PCB.Subsequent ligation mixture is purified using streptavidin interaction,for example, magnetic capture using Dynabead-280 streptavidin. Thecapture results in selective isolation of recombinant vector containingthe PCB-cassette and free PCB-labeled cassette. Photolysis releases themutant containing vector in pure form in any desired solution andconcentration. Subsequent transformants have very high probability ofcontaining only the desired mutant.

[0180] In current protocols, after restriction digestion of vector,complete purification of doubly restricted vector by, for example,agarose gel electrophoresis, is often difficult. The size difference ofthe resulting DNA fragments is typically very small. For example, thesize of a typical cassette is about 30 base-pairs (bp) and a typicalvector about 5000 bp. Restriction enzyme digestion to remove thecassette would produce fragments of 5000 bp and 30 bp which can bereadily distinguished and isolated. However, this is assuming thatcomplete digestion has occurred. Partial digestion would produce anadditional fragment of 5030 bp which is not easily detected much lessdistinguished or isolated. Thus, ineffective purification of restrictedvector creates a higher chance of ligation without incorporation of themutant cassette. Use of PCB avoids gel electrophoresis and subsequentelution of restricted fragments. Magnetic capture can be performed evenin the ligation mixture.

[0181] Liposomes are widely used for targeting and introduction ofbiologically important materials into cells via fusion into the cellmembrane (G. Gregoriadis editor, Liposome Technology, vol. III, CRCPress, Boca Raton, Fla., 1984). The avidin-biotin system is useful forin vitro studies to mediate between encapsulated liposomes and targetcells. These studies involve the use of biotin containing phospholipidsto introduce biotin into membranes (E. A. Bayer et al., Biochim.Biophys. Acta 550:464, 1979). Avidin-biotin system has also beenattempted for targeting drugs into cells. These studies have determinedthat the effects of biotinylation on the properties of the modifiedlipid and biotinylated molecule, albeit chemically altered, maintainstheir fundamental properties. For example, the biotinylated lipid isfully extractable in organic solvents, forms liposomes and mixedliposomes, and is correctly oriented in the latter (B. Rivnay et al.,Methods Enzymol. 149:119, 1987). Fatty acid components comprise theinterior of the bilayer and the biotinyl head groups are exposed to theaqueous environment of the solvent. Thus, the use of biotinylated lipidsfor liposome preparation allows an almost irreversible binding ofbiotin-streptavidin interaction. Although biotin and its interactionallows easy manipulation of these liposomes, any attempt to concentrateor separate these liposomes using immobilized streptavidin, such asmagnetic beads coated with streptavidin, fails as it is virtuallyimpossible to separate the concentrated/separated liposomes from boundstreptavidin.

[0182] Use of PCB-lipids readily overcomes these limitations asillumination releases the PCB-lipid containing liposomes into desiredsolutions at desired concentrations. The steps involved in themanipulations of liposomes using PCB-lipids include (1) attachment of aphotocleavable biotin derivative to a liposome by chemical means or,alternatively, incorporation of a photocleavable biotin lipid(PCB-lipid) into a liposome by first derivatizing the lipid with PCBfollowed by preparation of liposome (PCB-liposome), (2) concentration orseparation of the PCB-liposome through the selective interaction of thephotocleavable biotin with avidin, streptavidin or their derivativeswhich is normally immobilized on a material such as magnetic beads,affinity column packing materials and filters, and (3) detachment of thephotocleavable biotin from the PCB-liposome by illumination at awavelength which causes the photocleavable biotin covalent linkage to bebroken

[0183] The methods for attachment of various photocleavable biotinsdirectly to liposomes involves modification of the functional groups onthe lipids molecules using PCB. The choice of a particularphotocleavable biotin depends on which molecular groups are to bederivatized on the lipids constituting the liposome. For example,attachment of photocleavable biotin to a liposome could be accomplishedby forming a covalent bond with the amino group on thephosphatidylserine. Although a number of group-specific PCB derivativesare available that allow modification of any functional group to yieldPCB-lipid, phosphatidylethanolamine and phosphatidylserine areexemplary. PCB-phosphatidylserine and PCB-phosphatidylethanolamine areshown in FIG. 16.

[0184] Concentration and separation of liposome from a heterologousmixture is readily achieved using photocleavable biotin by establishedprocedures that are similar to the more conventional methods utilizingnon-cleavable biotin lipids. This normally involves an affinitytechnique based on streptavidin-biotin interaction whereby the liposomescontaining biotin are immobilized due to their interaction withstreptavidin. These techniques include streptavidin-coated magneticbeads, streptavidin-sepharose columns and streptavidin conjugatedfilters, all of which are commercially available. After concentratingPCB-liposomes using affinity interaction with streptavidin, liposomescan be released by illumination in desired solution and at the desiredconcentration.

[0185] Avidin-biotin technology is used extensively in the field ofaffinity cytochemistry where specific cell structures or subcellularcomponents are localized by selective labeling. Two different approachestermed immunohistochemistry (IHC) and in situ hybridization (ISH) areavailable. In IHC, a primary antibody or binding ligand which isbiotinylated (alternatively, a biotinylated secondary antibody can beutilized), is directed at a specific antigen on the surface of a cell orcellular structure. The cell or cellular structure is then localized byapplication of a reporter complex which could consist of anavidin-enzyme conjugate, avidin-fluorescence marker or avidin-ferritincomplex for electron microscopic localization.

[0186] In ISH, a DNA probe which is biotinylated is used to label thelocation of specific mRNA or DNA sequence in individual cells or tissuesections. A similar range of avidin based reporter complexes can be usedas in immunohistochemistry including enzyme, fluorescence and ferritinconjugated avidins. However, a serious limitation of the conventionalapplication of these two techniques is the difficulty of removing thebiotin-marker complex once a label has been applied to sample. Removalof the label would enable additional labels to be applied therebyproviding a means to map the interrelationship between various cellularand subcellular components in a tissue. The use of PCB provides aconvenient means to achieve this goal since photocleavage of the linkerconnecting the antibody or DNA probe and PCB will result in release ofthe marker complex.

[0187] In situ hybridization techniques are used to detect specificcellular DNA or which are non-uniformly distributed in individual cellsor tissue sections, and to detect viral nucleic acid sequences which areoften focal in distribution. In contrast to Southern-, Northern- ordot-blot hybridization assays which determine the average content oftarget molecules per cell in the extracted tissue, ISH detects specifictarget sequences that are focally distributed in a small number of cellsthat contain significant levels of target molecules (E. J. Gowans etal., Nucleic Acid Probes, R. H. Symons editor, CRC Press, Boca Raton,Fla., 1989).

[0188] Current technology is limited by fact that a single tissuesection, chromosome slide or cell-slide is usable only once and probingthe distribution of second target which could be another set ofsequences that are co-regulated or correlated, is almost impossible. PCBlabeled probes offer completely non-invasive approach for multiple insitu hybridizations on such valuable sample. Combined with multiple insitu hybridizations, a composite picture can be constructed ofdistribution of various nucleic acids. The protocol for ISH is adaptedfrom published procedures (D J. Brigati et al., Virol. 126:32, 1983; I.Guerin-Reverchon et al., J. Immunol. Meth. 123:167, 1989). The procedureinvolves careful fixation and sectioning of tissues which may be eitherparaffin or frozen sections. Fixation is designed not only to fixnucleic acids, but also to bind the section firmly to the slide.Glutaraldehyde is used for DNA and paraformaldehyde is used for RNAdetection. Both steps include optional denaturation steps which arenecessary if dsDNA or dsRNA is the target of reaction. Further stepsinvolve preparation of different probes that are labeled using PCB, andhybridization of these probes in a sequential manner. ISH follows thesame general principles as a solution and filter hybridization (R. J.Britton et al., Nucleic Acid Hybridization, B. D. Hames and S. J.Higgins editors, IRL Press, Oxford, 1985). PCB-labeled probe is thendetected using variety techniques that use streptavidin conjugateddetecting systems. A picture is obtained that shows distribution offirst probe in the tissue section or chromosome picture. Photolysisresults in release of detection assembly along with biotin moiety. ISHand detection is then repeated with second probe and its distribution isobtained. A composite picture is created that shows distribution of avariety of probes in the tissue section.

[0189] In situ hybridization (ISH) techniques are also used to detecteither specific cellular DNA or RNA sequences at the chromosomal levelin individual cells or tissue sections. The method is referred to ashybridization histochemistry. ISH is ideally suited not only to thedetection of cellular nucleic acid sequences which are non-uniformlydistributed in tissues or cell, but also to the detection of viralnucleic acid sequences which are often focal in distribution Thedetection of nucleic acid by ISH satisfies the primary objective ofreflecting accurately the intercellular and intracellular distributionof target molecules in the sample. In general, ISH may be used to detectDNA corresponding to normal or abnormal genes, to identify thechromosomal location of particular DNA sequences, and to measure thelevel of expression of these genes by mRNA detection

[0190] In particular, mRNA is a common target for in situ hybridizationreaction in studies of gene expression and cell differentiation In thespecial cases of virus infected cells, either viral genomic nucleic acidor RNA transcripts can be detected. ISH is especially valuable where thehistological mapping of target cells within a tissue is sought. Incontrast, Southern, Northern and dot-blot hybridization assays measurethe overall concentration of the target molecules per cell in theextracted tissue. If PCB is substituted for biotin in the application ofimmunochemistry and in situ hybridization, the methodology forlocalization of a label is almost identical. However, an importantadvantage is the ability to completely remove the label in the form of aPCB-avidin marker complex by simple illumination of the sample whichphotocleaves the PCB linkage to the antibody or hybridization probe.This step renders the sample, typically a tissue section, available forfurther sequential labeling by additional different probes.

[0191] There is often a need to determine if antigenic peptides,hormones or viral gene products detected intracellularly byimmunohistochemical techniques represent de novo synthesis or merelydeposition and passive accumulation It is also useful to determine ifspecific mRNA, detected by ISH is translated into protein product. Theuse of PCB facilitates such a determination since sequentialinterrogation of a single sample is possible using the sameenzyme-avidin linked reporter complex. This approach avoidscomplications due to the use of different samples.

[0192] Many different genes, difficult or impossible to locateotherwise, can be localized using conjugates of the invention.Neurotransmitter receptors are members of large gene families. By acombination of expression strategies and homology cloning, dozens ofreceptor genes in this family have now been cloned. These genes includethose encoding receptors for glutamate, glycine, and γ-aminobutyric acid(GABA) receptors. Cloning has revealed the existence of distinctneurotransmitter-receptors in numbers that had not been anticipated byneurophysiologists. The significance of this surprising receptorhomogeneity is not yet known but ISH has shown that individual receptorsubtypes are expressed in unique patterns in the brain. An importantgoal is to determine the distribution of these receptors in the brain.Ordinarily, this is done using many thin slices each exposed to adifferent hybridization probe. However, these experiments suffer fromthe use of multiple tissue sections. In contrast, the use of PCB wouldallow repeated analysis of the same rat brain section to determinecomplete distribution of each receptor expression.

[0193] Further, cloned genes and markers can be localized by ISH tospecific regions of chromosomes. Sequences can be localized tosub-chromosomal regions by hybridizing radioactively labeled probesdirectly to chromosome spreads. Chromosome spreads are made by usingcells whose division has been blocked in the metaphase by a chemicallike colcemid which disrupts the mitotic spindle. After fixing andstaining, a pattern of light and dark bands develops on each chromosome,so that the chromosomes can be identified. After ISH, location of theradioactive probe is revealed by the distribution of silver grains in aphotographic emulsion layered over the spread. However, the detection ofsingle copies of human genes is difficult and can be done only bypooling the distribution of grains over as many as 30 or more metaphasespreads.

[0194] PCB offers the unique advantage that the probe can bephotoreleased after localization of a specific genetic sequence on achromophore and a second probe applied. In cases where too closelyspaced sequences are to be detected, prior removal of the probe may beessential in order to avoid interference from the original probe. Inaddition, the method allows for the use of a large number of probes tobe applied sequentially and is not limited by the availability ofdifferent marker complexes which can be simultaneously measured. Thistechnique could also be used advantageously for rapid ordering ofmultiple probes on a chromosome.

[0195] An important application of biotin-avidin technology is theseparation of cells from a complex mixture often containing a variety ofdifferent cell types. Cells which can be utilized include cells within abiological sample, tissue culture cells, bacterial cells and diseasedcells. Cells may be mammalian, such as mammalian stem or fetal cells.Receptors include antibodies directed against the classical cell surfacereceptors and other surface proteins, but also any cell-surface moleculethat has a specific affinity for another molecule. Preferably, thereceptor is an antibody which recognizes a cell surface marker on thetarget cell or a specific protein which recognizes a cell surface ligandor other macromolecule on the target cell. PCB-modified antibodies,specific for surface antigens, receptors or ligands on the cell, can beused for cell separation or cell sorting with, for example, an apparatussuch as a fluorescence-activated cell sorter (FACS). ThesePCB-antibodies, when linked to streptavidin-coated magnetic beads canbind to the specific cell population bearing a particular antigen andcan then be separated using ImmunoMagnetic Separation (IMS).

[0196] Current methodologies do not allow gentle separation of thesemagnetic particles from separated or sorted cell population. PCBmodified antibodies, however, can be readily separated from magneticparticle after illumination. IMS involving PCB-antibodies can be used incell-sorting, tissue typing and for selective enrichment ofmicroorganisms using modifications of protocols described earlier (A.Elbe, J. Immunol. 149:1694, 1992; S. Qin, Sci. 259:974, 1993; L.Leclerecq, Immunol. Lett. 28:135, 1991).

[0197] In conventional methods, a biotinylated antibody is utilizedwhich binds selectively to an antigen residing only on the target cell.The target cells can then be isolated from the mixture by usingstreptavidin-coated magnetic beads or streptavidin based affinitycolumns. The affinity material sometimes contains a secondary antibodydirected toward the primary antibody. A severe limitation of thisapproach is the difficulty of releasing the cells once they are bound tothe affinity medium through the biotin-streptavidin interaction. Normalmethods that are designed to disrupt the biotin-streptavidin interactionor the antibody-antigen interaction can reduce the overall viability ofthe released cells. This is a serious disadvantage if the cells are tobe used later for culturing or transplantation. Similarly, conditionssuch as low pH that disrupt the antibody-antigen interaction can causecell damage. A standard technique is overnight incubation in a culturemedium followed by vigorous mixing. This causes shedding of the antigeninvolved in binding. However, this method is time consuming, can lead tocell degradation and does not result in complete release. An alternatemethod is to disrupt the antibody-antigen interaction with enzymatictreatment, which can also be damaging to a cell. An additional method isthe utilization of anti-FAB antibodies to compete for the antigen. Suchmethods are time consuming, expensive due to the use of antibodies andonly partially effective. These methods of detachment (release) of cellsare all particularly ineffective when the antibody-antigen interactionis strong or the binding involves several antigen-antibody interactionsmixing. Conventional biotins which can be chemically cleaved such asNHS-SS-Biotin (sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate; Pierce Chemical; Rockford, Ill.) poseserious problems since the cleavage medium consisting of a highconcentration of reducing agents such as thiols will cause reduction ofprotein disulfide bonds and a subsequent loss of cell viability.

[0198] In contrast to conventional methods, photocleavable biotin offersan inexpensive, effective and rapid means to release immobilized cellssimply by using light exposure. Release occurs due to photocleavage ofthe covalent linkage between the antibody and biotin. While the antibodystill remains bound on the released cell, this is normally not a problemfor cell integrity or additional utilization of the separated cells.Since photocleavage can be performed in short periods and results inalmost full removal of the photocleavable biotin release is rapid andcomplete.

[0199] Further, using photocleavable conjugates, cells which represent avery small populations of sample of cells can be accurately andefficiently selected. This enables methods such as the selection ofimmune cells, stem cells, fetal cells, precursor cells and nearly andcell type from lymph (e.g. interstitial, lymphatic), blood (e.g.arterial and peripheral blood) or tissue (organ, soft tissues, muscle)samples. Selected and isolated cells can then be cultured in very largenumbers and possible reintroduced into the same or another patient.Cultured cells can also be used in gene therapy by the introduction ofgenetic material into cultured cells. Such techniques are not possibleusing conventional detection and isolation procedures.

[0200] The same approach can also be used by linking photocleavablebiotin to other cell specific macromolecules such as cell-associatedligands or antigens. For example, B cells expressing a specificimmunoglobulin receptor for a target antigen could be isolated byattaching photocleavable biotin to the target antigen and then bindingit to a streptavidin-coated bead. Alternatively hybridoma screening andstem cell selection could be performed using this approach.

[0201] The three basic steps involved in cell isolation using PCB are(1) attachment of a photocleavable biotin, or molecule containing aphotocleavable biotin derivative including (antibodies, receptorligands, antigen) to the surface of the target cell by a photochemicallycleavable bond, (2) separation of said cell type from other cells andmaterials in the complex mixture through the selective interaction ofthe photocleavable biotin with avidin, streptavidin or theirderivatives, and (3) detachment of the photocleavable biotin from thesaid cell type by illumination at a wavelength which causes thephotocleavable biotin covalent linkage to be broken. These steps, usingPCB, can be carried out in combination with ordinary biotin. In thiscase, cells containing two or more surface markers can be from thosecontaining only one. Each of these steps is illustrated in FIG. 17.

[0202] Several applications for the use of PCB for cell separationoffers clear advantages over existing technology. A common method forisolation of cells is to label the cell with a fluorescent dye which isdirected to the target cell through an antibody-fluorescent conjugate.The cells can than be separated by using a fluorescence-activated cellsorting device and used for a variety of purposes including cell typing,hybridoma production and tissue culturing. However, with this techniquethe separated cells remain labeled with the fluorescent dye. This canresult in reduction in cell viability, prevent their use in therapeuticapplications where the dye can be toxic, and prevent furtherfluorescence based sorting into subspecies of cells. One example wouldbe the separation of lymphocytes using characteristic antigens such asCD2, CD4, CD8 and CD19 followed by sorting into subspecies.

[0203] An alternative approach is to utilize an antibody which isconjugated to PCB. In this case the cell can be fluorescence labeledwith an avidin-fluorescein complex which is commercially available in avariety of forms (Table 8). The fluorescent label can then be removed byphotocleavage of the PCB which results in release of thePCB-avidin-fluorescent complex. A variety of fluorescent labels existwhich absorb in a region outside of the absorption band of the PCB, thusavoiding photocleavage during the cell sorting. TABLE 8 CommerciallyAvailable Avidin-Florescent Dye Complexes Conjugate Absorption (nm)Emission Producer Avidin- 490 520 Pierce Fluorescein Avidin-R- 450-470574 Pierce Phycoerythrin Avidin- 515-520 575 Pierce Rhodamine Avidin-575 600 Pierce Rhodamine 600 Avidin-Texas 595 615 Pierce Red

[0204] Another example of the utility of PCB methodology for cellisolation is the isolation of B cells for hybridoma production Briefly,formation of hybridomas for the purpose of monoclonal antibodyproduction involves the fusion of myeloma cells with lymphocyte B cells.Generally, a heterogeneous population of B cells is utilized whichcontains different immunoglobulin receptors. The hybridoma whichexpresses the desired antibody is then screened by assaying for bindingto a particular antigen. This screening process can be time-consumingand expensive. Screening could be avoided if a method existed forisolation of a particular population of B cells which only expressed thedesired immunoglobulin receptor. In principle, this could beaccomplished by using a biotinylated antigen, such as a polypeptide witha specific sequence, which will only bind to those B cells which expressthe specific immunoglobulin receptors for the antigen. Thissubpopulation of B cells could then be selected by using avidin affinitytechniques such avidin-coated magnetic beads. However, the subsequentstep of hybridoma formation will still be prevented unless the B cellscan be released from the avidin-biotin complex in a viable form.

[0205] The use of PCB-biotin avoids this problem by providing a simplemethod for releasing the B cells in a viable form after immobilizationby the biotin-avidin interaction. Photocleavage of the photocleavablebiotin linkage with the antibody results in the release of the B-cellsand the bound antigen which is now in an unmodified form Since nochemical treatment is required, the cells will retain their viabilityfor further fusion to the myeloma cells. Furthermore, the method israpid and suitable for automation.

[0206] Another embodiment of the invention is directed to targetsisolated by the above method which may be utilized in pharmaceuticalcompositions to treat or prevent diseases and disorders. Pharmaceuticalcompositions may comprise the isolated targets plus a pharmaceuticallyacceptable carrier such as water, oils, lipids, saccharides,polysaccharides, glycerols, collagens or combinations of thesecomponents. The composition is administered to patients for thetreatment or prevention of certain diseases and disorders and for thesite-directed administration of pharmaceutical agents.

[0207] Another embodiment of the invention is directed to a method fordetermining an in vivo half-life of a pharmaceutical in a patient.Conjugates are formed by coupling the pharmaceutical to a bioreactiveagent via a covalent bond that can be selectively cleaved withelectromagnetic radiation. Conjugates are administered to the patientafter which, two or more biological samples are removed. Samples aretreated with electromagnetic radiation to release the pharmaceuticalfrom the bioreactive agent, the amount of the bioreactive agent in thebiological samples is determined, and the in vivo half-life of thepharmaceutical determined.

[0208] The pharmaceutical may be a composition comprising cytokines,immune system modulators, agents of the hematopoietic system,chemotherapeutic agents, radio-isotopes, antigens, anti-neoplasticagents, recombinant proteins, enzymes, PCR products, nucleic acids,hormones, vaccines, haptens, toxins, antibiotics, nascent proteins,synthetic and recombinant pharmaceuticals, and derivatives andcombinations of these components. Conjugates may be administered topatients by parenteral administration, sublingual administration,enteral administration, pulmonary absorption, topical application andcombinations thereof. Animals which can be tested include mammals suchas humans, cattle, pigs, sheep, dogs, cats, horses and rodents.Biological samples which are collected can be sample of peripheralblood, blood plasma, serum, cerebrospinal fluid, lymph, urine, stool,ophthalmic fluids, organs and bodily tissues.

[0209] Another embodiment of the invention is directed to the controlledrelease of a substrate into a medium. Conjugates comprised of abioreactive agent coupled to the substrate by a covalent bond which canbe selectively cleaved with electromagnetic radiation are created asdescribed. These conjugates are bound to a surface of an article andplaced into the medium The surface of the article is exposed to ameasured amount of electromagnetic radiation and the substrate releasedinto the medium to carry out a beneficial effect. Alternatively, anarticle may be placed at a selected site and the conjugates, having anaffinity for the article, are administered at a distal site. Conjugatesthen migrate to the selected site and perform an intended function.After completion of that function, radiation is applied and thesubstrate is released from the fixed bioreactive agents. Releasedsubstrate may be naturally eliminated from the patient's system. Thiscan be highly useful, for example, in radiation therapy for cancerpatients.

[0210] Preferred are radiation wavelengths which can penetrate themedium. Depending on the amount and frequency of radiation exposure,release can be controlled and continued over a period of time. Thismethod is useful for the controlled and site-directed administration ofpharmaceutical compositions to a patient. In such cases, the medium inwhich the article is placed may be blood, lymph, interstitial fluid or atissue. Controlled release may also be performed in tissue culture foradministering a constant or periodic amount of a substrate to a cellculture fluid or balanced salt solution for uptake by the cells.Articles which may be coupled with substrate and placed within oradjacent to a patient's body include articles comprising carbohydrates,lipids, proteins, polysaccharides, cellulose, metals including magnets,organic polymers and combinations thereof. Preferably, the surface ofthe article is coated with streptavidin and the bioreactive agent isphotocleavable biotin.

[0211] Alternatively, articles containing conjugates or agents can beplaced into the site of the disorder, such as a tumor. Thepharmaceutical agent such as, for example, a radioactive agent isadministered to the patient and becomes bound to the fixed conjugates oragents. Effects attributable to the pharmaceutical agent are localized.The article is exposed to a measured amount of electromagnetic radiationand the pharmaceutical agents released into the body and excreted. Thismethod is preferred when only a short term exposure of thepharmaceutical agent is desired or to efficiently remove potentiallyharmful agents after they have had their desired effects.

[0212] Another embodiment of the invention is directed to a method forcreating a photocleavable oligonucleotide. A bioreactive agent iscreated comprised of a photoreactive moiety coupled to a detectablemoiety and containing a phosphoramidite. The oligonucleotide issynthesized using conventional phosphoramidite chemistry. The nucleotideprecursors comprise one or more photocleavable phosphoramidites such aspurine-phosphoramidites (uracil, cytosine, thymine) orpyrimidine-phosphoramidites (adenine, guanine) as the ribose ordeoxyribose forms, or derivatives thereof This method can be performedmanually or automated using a commercially available oligonucleotidesynthesizer. Photocleavable oligonucleotides can be utilized as primersor probes, in diagnostic kits and in every instance in which a nucleicacid can be used.

[0213] Another embodiment of the invention is directed to a method fordetecting a target molecule in a heterologous mixture. Conjugates areformed by coupling a substrate to a bioreactive agent with a covalentbond that is selectively cleavable with electromagnetic radiationConjugates are contacted with the heterologous mixture to couplesubstrate to one or more target molecules. Uncoupled conjugates areremoved and the coupled conjugate are treated with electromagneticradiation to release the detectable moiety. Presence of target moleculescan be detected by detecting the presence of the released detectablemoiety. Target macromolecules may be proteins, peptides, nucleic acids,lipids, polysaccharides, metallic compounds, virus, bacteria, eukaryoticcells, parasites and derivatives and combinations thereof. Oncedetected, target macromolecules can be isolated and the amount isolatedquantitated by current any of the techniques available to those ofordinary skill in the art.

[0214] A specific application of streptavidin-biotin technology is inthe detection of targets in medical diagnosis. Generally, the target isa biotinylated molecule or a biotinylated probe for the target molecule.The interaction of streptavidin with the biotinylated target or probe isamplified by an enzyme conjugated to streptavidin which catalyzes achromogenic reaction. For example, a variety of enzymes conjugated tostreptavidin are commercially available including horseradishperoxidase, β-galactosidase, glucose oxidase and alkaline phosphate.Each of these enzymes catalyze a chromogenic reaction. Additionalmethods of detection include conjugating streptavidin to fluorescent,chemi-fluorescent, radioactive or electron dense molecules.

[0215] An example of biotin-avidin interaction in medical diagnosis alsoforms the basis for a wide array of enzyme-linked immunospecific assays(ELISA). In this case, an antibody specific for the target molecule, theprimary antibody, or a secondary antibody directed against the primaryantibody is biotinylated. Detection is accomplished with astreptavidin-enzyme conjugate as described. A large number ofimmunoassays have been developed based on this approach. However,multiple immunoassays on the same sample are not easily accomplishedusing conventional technology since there exists no simple method ofremoving the avidin-enzyme-complex once bound to a biotin derivativewithout damaging the sample. In contrast to traditional methodologies,PCB can be reused many times for multiple assays of the same sample formultiple screening of pathogens and other markers of human diseases.

[0216] The biotin-avidin interaction also forms the basis of sensitivemethods for detecting specific nucleic acid sequences such as screeninghuman DNA samples. DNA or RNA probes which hybridize to a target DNAsequence are biotinylated. DNA containing the target sequence isdetected with an streptavidin-enzyme conjugate. This method is used inconjunction with the polymerase chain reaction where the target gene orsequence to be screened is first amplified using specific primers. Theintroduction of biotin nucleotides into the primer or directly into thenascent PCR product using biotinylated nucleotides facilities isolationof the target DNA. A variety of biotinylated nucleotides such asbiotin-dUTP are available for such purposes. However, this method has atleast two limitations.

[0217] First, the presence of biotin in the amplified target DNAprevents the use of biotinylated probes without prior removal of thebiotinylation. The presence of endogenous biotin or biotin-containingmolecules in the sample also lowers the sensitivity of this assay.Second, the presence of biotin in the target DNA lowers hybridizationefficiency and hence the sensitivity of the assay. As discussed above,the use of PCB derivatives such as PCB-nucleotides effectivelyeliminates these problems by allowing for complete removal of biotinfrom the procedure.

[0218] Conjugates and methods of the invention can be used inconjunction with a variety of diagnostic assay involving nascent proteindetection. For example, diagnostic assays for cancer have been developedwhich rely on in vitro expression of PCR amplified genes followed byexamination of the nascent protein product using gel electrophoresis (S.M. Powell et al., N. Engl. J. Med. 329:1982, 1993). The isolation ofsuch proteins and the subsequent sensitivity of such tests could beincreased by the incorporation of PCB amino acids.

[0219] Biotin-streptavidin technology is widely used as the basis fornon-radioactive ELISA including diagnostic assays for specificindicators of diseases and disorders such as disease-linked antigensincluding adenovirus antigen (K Mortensson-Egnund et al., J. Virol.Methods 14:57, 1986), bovine leukemia virus (E. N. Esteban et al.,Cancer Res. 45:3231, 1985), flavivirus (E. A. Gould et al., J. Virol.Methods 11:41, 1985), Hepatitis B surface antigen (C. Kendall et al., J.Immol. Methods 56:329, 1983), Herpes simplex virus antigen (K.Adler-Strorthz et al., J. Clin. Microbiol. 18:1329, 1983), respiratorysyncytial virus (A. Hornsleth et al., J. Med. Virol. 18:113, 1986),bacterial antigens (R. H. Yolken et al., J. Immunol. Methods 56:319,1983) and melanoma-associated antigens (human) (AC. Morgan et al.,Cancer Res. 43:3155, 1983). The usefulness of these assays can becompromised if endogenous biotin is present in the sample. In this case,a false background will be obtained since the streptavidin-reporterenzyme complex will react both to non-specific biotins and to thebiotinylated antibodies directed against the target protein. Whileseveral approaches to eliminate background due to non-specific bindingof the avidin or streptavidin to non-biotinylated targets including theuse of high ionic-strength. buffers (C. J. P. Jones et al., Histochem.J. 19:264, 1987), milk proteins (R. C. Duhamel et al., J. Histochem.Cytochem. 33:711, 1985) and lysozyme (E. A. Bayer et al., Anal. Biochem.163:204, 1987) and altered streptavidins such as ImmunoPure NeutrAvidin(Pierce Chemical; Rockford, Ill.), none has been very effective ineliminating background due to endogenous biotin.

[0220] Use of photocleavable biotins in conjunction with immunoassaysalleviates the problem by providing a means of determining thebackground level of non-specific biotin. The ELISA assay is firstperformed using conventional methodology except that the antibodydirected against the protein is targeted with photocleavable biotin(FIG. 19). The streptavidin-reporter enzyme complex linked to the probeantibody system is then removed via light cleavage of the photocleavablebiotin. Cleavage does not release the reporter enzyme-avidin complexbound from non-specific bound biotin since this biotin is non-cleavable.The signal obtained from the remaining biotin can be used as a measureof the endogenous biotin present in the sample and subtracted from theprimary signal obtained in step 1.

[0221] Background signal due to endogenous biotin in a biotin-avidinbased ELISA can be simply detected using photocleavable biotin. TheELISA is performed according to normal procedures usingstreptavidin-reporter enzyme complex. However, the streptavidin-reporterenzyme complex is then removed with light and the system reassayed todetermine the background level of endogenous biotin.

[0222] A second application of photocleavable biotins is its use toconduct multiple biotin based ELISA assays on the same sample. This. isbased on the ability to fully remove the streptavidin-reporter enzymecomplex with light upon photocleavage of the PCB linkage. In this case asecond ELISA can-be performed using a different antibody probe andreporter enzyme complex without interference from the first ELISA asshown in FIG. 20. Normally such a multiple ELISA is not possible becausethe signal obtained from a second antibody probe combined with astreptavidin-reporter enzyme complex cannot be easily separated from theoriginal signal.

[0223] For example, in a conventional biotin-avidin ELISA, thestreptavidin-reporter enzyme complex remains bound to the probe antibodyeven after the chromogenic product of the reporter enzyme is removed.Thus, it will continue to produce a chromogenic product even after asecond ELISA is performed and interfere with the signal of that secondimmunoassay. In contrast, the removal of the streptavidin-enzyme complexby cleavage of the photocleavable biotin with light and then subsequentwashes eliminates this problem since the original reporter enzyme is nolonger present.

[0224] In another application of the invention, detection of pathogenssuch as microorganisms from biological material often requires theirisolation and culturing. The more effective the isolation step, the morereliable and rapid the culturing step will be because of the eliminationof other contaminants and the concentration of the target pathogen.While a variety of affinity techniques exists for separation ofmicroorganisms such as magnetic beads conjugated to selectiveantibodies, the problem of release of the microorganisms in a viableform suitable for culturing and sensitive detection still remains. Incontrast, PCB which is linked to the antibody or binding ligand providesa non-damaging and rapid means for photochemical release of themicroorganism in a viable form.

[0225] For example, this application of the invention provides the basisfor development of rapid diagnostic assays for a variety of pathogensinvolved in human and animal disease that were previously not possibleusing conventional biotin-streptavidin technology. Microorganisms couldalso be isolated from food, milk, soil and other materials for thepurpose of depletion or detection using this approach.

[0226] Another embodiment of the invention is directed to methods fortreating a disorder by the controlled release of a therapeutic agent ata selected site. Conjugates are formed by binding a bioreactive agent toa therapeutic agent with a bond that is selectively cleavable withelectromagnetic radiation, wherein the bioreactive agent is comprised ofa directable moiety bonded to a photoreactive moiety and the directablemoiety has an affinity for the selected site. Conjugates areadministered to a patient having the disorder and the selected site issubjected to a measured amount of electromagnetic radiation for thecontrolled release of the therapeutic agent to treat the disorder.Disorders which can be detected include infections, such as bacterialinfections, viral infections and parasitic infections, neoplasias suchas a tumor, and genetic disorders such as an overproduction ordeficiency of an enzyme or other genetic product.

[0227] The therapeutic agents may be toxins, immune system modulators,hematopoietic agents, proteins, nucleic acids, substrate analogstranscription and translation factors, antigens and combinationsthereof. Directable moieties may be antibodies such as a monoclonal orpolyclonal antibody or antibody fragment.

[0228] Another embodiment of the invention is directed to diagnostickits for detecting or screening for diseases and disorders in patients.Kits contain a conjugate comprised of a bioreactive agent, covalentlybonded to a diagnostic agent having an affinity for an indicator of saiddisorder in a biological sample obtained from the patient. The indicatormay be a presence or absence or an increased or decreased amount orlevel of a characteristic marker of the disorder such as an antigen orantibody, a cytokine, a specific cell type (e.g. B cells; cytotoxic,suppressor or helper T cells; macrophages; stem cells), a particularenzyme, nucleic acid or protein. Disorders which can be detected includeinfections, neoplasias and genetic disorders. Infections which can bedetected include bacterial infections, viral infections and parasiticinfections. Neoplasias which can be detected include tumors. Geneticdisorders which can be detected include an overproduction or deficiencyof an enzyme. Biological samples which can be added to the sampleinclude samples of peripheral blood, blood plasma, serum, cerebrospinalfluid, lymph, urine, stool, ophthalmic fluids, organs and bodilytissues. Such kits may also be used to detect or screen for the presenceof fetal or stem cells in a biological sample which can be isolated andcultured or further analyzed.

[0229] Kits may also be used to detect the presence of multiple nucleicacids and/or proteins on, for example, an electroblot using a series ofsecondary probes linked to biotin. After each probe is introduced, thebiotin attachment could be cleaved allowing the enzymatic assay complexto be removed thus providing for a new secondary probe to be introduced.Such an approach would be extremely useful as the basis of medicaldiagnostic assays, where multiple antigens or nucleic acid sequencesneeded to be probed rapidly and automatically.

[0230] The kit may also be a nucleic acid mutagenesis kit for use inmolecular biological applications such as introducing or correctingmutations in DNA or RNA. The nucleic acid may be an oligonucleotide foruse in PCR or cassette-type applications. Such oligonucleotides may besingle-stranded or double-stranded and preferably contain one or morerestriction enzyme recognition sequences internally and ligatable 5′ and3′ ends which also contain part of a restriction enzyme recognitionsite. Alternatively, one or more ends may be blocked to facilitatedirected coupling.

[0231] The following examples are offered to illustrate variousembodiments of the invention, but should not be viewed as limiting thescope of the invention.

EXAMPLES Example 1 Synthesis of Photocleavable Agents

[0232] Five grams or 27.6 mmol of 5-methyl-2-nitrobenzoic acid (FIG. 5,compound “6”; Aldrich Chemical; Milwaukee, Wis.) was added in smallportions to 10 ml (16.4 g or 148 mmol) of thionyl chloride withstirring. The mixture was stirred at room temperature for 10 hours.Excess of thionyl chloride was removed by vacuum to give the acidchloride (“7”). Magnesium turnings (1.07 g or 44.2 mmol), absoluteethanol (6 ml), chlorobenzene (8 ml), and 0.1 ml of dry CCl₄, wererefluxed until most of the magnesium reacted. A solution of diethylmalonate (4.82 g or mmol) in 10 ml of chlorobenzene was added followedby the addition of the acid chloride (5.49 g) in 10 ml of chlorobenzene.The reaction mixture stirred for 1 hour and 1.7 ml of concentrated H₂SO₄in 17 ml of H₂O was added, stirred for additional 15 minutes. 20 ml ofchloroform was added and the layers separated. The aqueous layer wasextracted three times with 10 ml and the extracts were combined, driedand evaporated to dryness. Residue was dissolved in 8.25 mls of aceticacid. 5.4 ml of H₂O and 1 ml of concentrated H₂SO₄ were added, themixture was refluxed for 6 hours, neutralized with aqueous Na₂CO₃ andextracted three times with 20 ml of CHCl₃. Extracts were combined, driedand solvents removed by vacuum. Residue was crystallized from 70%ethanol to produce 4.46 g, or about 81%, of5-methyl-2-nitroacetophenone. 5-methyl-2-nitroacetophenone (3.51 g or19.6 mmol), N-bromosuccinimide (3.66 g or 20.6 mmol), and benzoylperoxide (46 mg or 0.01 eq) were refluxed in 20 ml of CCl, for 5 hours.The reaction mixture was filtered, the filtrate concentrated andcrystallized from CCl₄ to produce 3.64 g (72%) of5-bromomethyl-2-nitroacetophenone (“8”). Compound 8 (2.0 g or 7.75 mmol)was added to a solution of hexamethylenetetramine (1.14 g or 8.13 mmol)in 15 ml of chlorobenzene. The mixture was stirred overnight, theprecipitate filtered off and washed with 10 mls of chlorobenzene and 20mls of diethyl ether. The precipitate (2.93 g or 736 mmol) was suspendedin 35 ml of 95% ethanol followed by the addition of concentrated HCl(3.12 ml or 5 eq.). The mixture was stirred overnight and evaporated todryness. Ten mls of DMF were added to the residue followed by theaddition of a 6-biotinamidocaproic acid (3.29 g or 1.25 eq.) in 35 ml ofN-methylpyrrolidone, dicyclohexylcarbodiimide (228 g or 1.5 eq.), andtriethylamine (1.28 ml or 1.25 eq.). The solution was stirred overnightat room temperature, the precipitate filtered off, and filtrateprecipitated to 700 ml of diethyl ether. The precipitate was dried andpurified on a silica gel column using step gradient (5-20%) of MeOH inCHCl₃ to produce 2.27 g (about 58%) of compound 11.

[0233] Compound 11 (1 g or 1.87 mmol) was dissolved in 15 ml of 70% EtOH(FIG. 5). The solution was cooled to 0° C. and sodium borohydride (141mg or 4 eq.) added. The solution was stirred at 0° C. for 30 minutes andat room temperature for an additional 2 hours. The reaction was quenchedwith the addition of 1 ml acetone, neutralized with 0.1N HCl,concentrated, the supernatant discarded, the residue washed with waterand dried to produce 0.71 g (about 71%) of compound 12.

[0234] Compound 12 (1.07 g or 2 mmol) was dissolved in 10 ml DMF.N,N′-disuccinimidyl carbonate (Fluka Chemical; Ronkonkoma, N.Y.) (1 g,1.5 eq.) was added followed by 0.081 ml or 3 eq. of triethylamine (FIG.5). After 5 hours at room temperature, solvents were evaporated todryness and the residue was washed consecutively with 0.1N NaHCO₃,water, dioxane, diethyl ether and dried to give 1.04 g (about 69%) of5-(5-biotinamidocaproamidomethyl)-1-(2-nitro)phenylethyl-N-hydroxysuccinimidyl carbonate (PCB-NHS) ester (compound13). M.P.=113-114° C. (uncorrected); CI-MS (M⁺=676.5); UV-VISλ=204 nm,ε1=19190 M⁻¹ cm⁻¹; λ2=272 nm, ε2=6350 M⁻¹ cm⁻¹ in phosphate buffer,pH=7.4. ¹H NMR (DMSO-d₆, Varian XL-400 MHz), [δppm]: 8.48 (t, 1H),8.05-8.03 (d, 1H), 7.75-7.71 (t, 1H), 7.66 (s, 1H), 7.46-7.45 (d, 1H),6.44 (s, 1H), 6.37 (s, 1H), 6.28-6.27 (m, 1H), 439 (m, 2H), 4.30 (r,1H), 4.12 (m, 1H), 357 (d, 2H), 3.09 (m, 1H), 3.01-2.99 (m, 2H), 2.79(m, 5H), 2.58-255 (d, 1H), 2.17-2.15 (m, 2H), 2.04-2.02 (m, 2H),1.72-1.71 (m, 2H), 1.66-1.43 (m, br, 6H), 138-136 (m, br, 2H), 1.26-1.25(m, br, 3H); IR (KBr); Vc=o 1815 and 1790 cm⁻¹.

[0235] Synthesis of PCB-NHS Ester (FIG. 18):

[0236] 2-Bromo-2′-nitroacetophenone (“14”) (Aldrich Chemical; Milwaukee,Wis.) (1 g; 4.09 mmol) was converted into 2-amino-2′-nitroacetophenonehydrochloride (“15”) by reaction with 1.05 eq. of hexamethylenetetramineand hydrolysis, and was coupled to 5-biotinamidocaproic acid (1.25 eq.)using DCC (1.5 eq.) in DMF to produce2-(5-biotinamidocaproamido)-2′-nitroaceptophenone (“16”) (about 52%yield) which was reduced (about 75% yield; “17” and converted intoreactive NHS derivative (“18”)2-(5-biotinamidocaproamido)-2′-nitrophenylethyl-N-hydroxysuccinimidylcarbonate (about 69% yield) as described.

[0237] Synthesis of PCB-NHS Ester (FIG. 19):

[0238] 3-amino-4-methoxybenzoic acid (“19”) (Aldrich Chemical;Milwaukee, Wis.) (5 g or 29.9 mmol) was suspended in 40 ml acetic acid.Acetic anhydride (3 ml or 1:04 eq) was added by stirring. The reactionmixture was stirred for 2 hours at room temperature. 25 ml of 0.1N HClwas added and the precipitate was filtered off and washed with 3×10 mlof 0.1N HCl and 5×10 ml water to produce 5.97 g (about 95%).3-(N-acetyl) amino-4-methoxybenzoic acid (“20”) (5 g or 23.5 mmol) wasadded to 20 ml of fuming nitric acid at 0° C. on vigorous stirring. Thesolution was stirred at 0° C. for an additional hour and poured onto 200g of ice. Precipitate was filtered off, washed with 5×20 mls of waterand dried to produce 438 g (about 72%) of 2-nitro-4-methoxy-5(N-acetyl)aminobenzoic acid (“21”), which was converted into2-nitro-4-methoxy-5-(N-acetyl)aminoacetophenone (“22”) (about 63% yield)as described. 2-nitro-4-methoxy-5-(N-acetyl)aminoacetophenone (“22”) (1g or 4,76 mmol) was dissolved in 5 ml of DMF and the solution was addedto 5-biotinamidocaproyl chloride prepared separately from5-biotinamidocaproic acid (1.55 g or 1 eq.) and 5 ml thionyl chloride.The reaction mixture was stirred overnight at room temperature and addedto 100 ml of diethyl ether. Precipitate was purified using small silicagel column and a step gradient of MeOH in CHCl₃ to give 1.16 g (about47%) of 2-nitro-4-methoxy-5-(5-biotinamidocaproamido) acetophenone(“23”). This intermediate was converted into its corresponding alcohol(about 85% yield) and into the reactive NHS ester derivative5-(5-biotinamidocaproamido)4methoxy-1-(2-nitro)phenylethyl-N-hydroxysuccinimidylcarbonate (“25”) with about a 69% yield.

[0239] Synthesis of Photocleavable Coumarin (FIG. 21):

[0240] 5-aminomethyl-2-nitroacetophenone hydrochloride (“26”) (1.15 g or5 mmol) was dissolved in 20 ml DMF. To this solution7-Methoxycoumarin-4-acetic acid (“27”) (Aldrich Chemical; Milwaukee,Wis.) (1.46 g or 1.25 eq.) and dicyclohexylcarbodiimide (1.55 g or 1.5eq.) was added followed by triethylamine (0.7 ml or 1 eq.). The solutionwas stirred overnight at room temperature, 20 ml of CHCl₃ was added, thesolution was washed with 0.1N NaHCO₃ (3×15 ml), and the organic layerwas dried and purified on a silica gel column using step gradient ofMeOH in CH₂Cl₂ to give 1.37 g (about 64%) of (“28”). Compound 28 (1 g or2.33 mmol) was dissolved in 15 ml of 95% EtOH, the solution cooled to 0°C. and sodium borohydride (176 mg or 4 eq.) added. The solution wasstirred at 0° C. for 1 hour and the reaction was quenched by addition of1 ml acetone and neutralized with 0.1N HCl. The solution was thenextracted with 3×15 ml of CHCl₃. Extracts were combined, dried, andpurified on a silica gel column using step gradient of MeOH in CH₂Cl₂ togive 832 mg (about 83%) of compound 29. Compound 29 (1.29 g or 3 mmol)was dissolved in 10 ml of DMF-acetonitrile (1:1). N,N′-disuccinimidylcarbonate (Fluka Chemical; Ronkonkoma, N.Y.) (1.15 g or 15 eq.) wasadded which was followed with chloroform. The solution was washed with3×15 ml of 0.1N NaHCO₃, solvents were evaporated to dryness and theresidue recrystallized from acetonitrile to give 1.22 g (about 71%) ofphotocleavable coumarin NHS ester (“30”).

Example 2 Synthesis of Photocleavable Conjugates—PCB-Amino Acids

[0241] PCB-amino acids were prepared by derivatization of the α-aminogroup of the amino acid. Derivatives were prepared from either PCBchloroformates or their corresponding N-hydroxysuccinimidyl esters andamino acids in a weakly alkaline media using a modification of theprocedure by Sigler et al. (Biopolymers 22:2157, 1983; L. Lapatsanis etal., Can. J. Chem. 60:976, 1982; A Paquet, Can. J. Chem. 60:2711, 1977).This procedure was also used for the synthesis of ε-NH₂-PCB-Lys whereinthe α-amino group of Lys is protected with Fmoc. PCB-amino acids werealso prepared by carboxyl or hydroxy group derivatization. Briefly,carboxyl residues of aspartic acid and glutamic acid were esterifiedusing PCB-OH. α-amino groups were protected as Fmoc derivatives andα-carboxyl groups were protected as t-butyl esters. Esterification ofthe carboxyl side chain was mediated using dicyclohexylcarbodiimide(DCC) (P. Sieber, Helv. Chim Acta 60:2711, 1977). Esterification wasalso carried out by reacting PCB-chloroformate with the hydroxyl sidechains of appropriately protected threonine or serine residues (M.Bednarek et al., Int. J. Pept. Prot. Res. 21:196, 1983; H. Kessler etal., Tetrahedron 281, 1983).

[0242] Preparation and Photocleavage of PCB-Leucine-Enkephaline:

[0243] Leucine-enkephaline (Sigma Chemical; St. Louis, Mo.) (15.5μmol/ml in 0.1N NaHCO₃, pH 8.0) and PCB-NHS ester (17 μmol/ml in DMF)were mixed and stirred overnight at room temperature. At this time HPLCanalysis showed complete conversion of Leu-Enk into PCB-Leu-Enk. HPLCanalysis was performed on a Waters System (Waters Chromatography;Marlboro, Mass.) comprising of U6K injector, 600 Controller, NovapakC₁₀-Column (3.9×150 mm) and 996 Photodiode Array detector. Tests wereperformed using a linear gradient 30-45% of B over 10 minutes followedby 45% of B isocratic for 10 minutes.

[0244] PCB-Leu Enk (1.93 μmol/ml in phosphate buffer, pH 7.4) wasirradiated with a long-wavelength, UV-lamp (Blak Ray XX-15 UV lamp; UVPInc; San Gabriel, Calif.) at a distance of 15 cm (emission peak 365 nm,lamp intensity=1.1 mW/cM² at a distance of 31 cm). HPLC analysis showedcomplete photorelease of Leu-Enk within 5 minutes and confirmedauthenticity of the released material on the basis of retention time andUV spectra.

[0245] PCB-Leu-Enk (10 nmoles) was incubated for 30 minutes in asuspension of monomeric-avidin agarose beads (15 nmoles). The suspensionwas spun-filtered for 3 minutes (16,000×). Binding efficiency wasdetermined at about 94%. Sample was resuspended in phosphate buffer (2ml) and irradiated as described. Released Leu-Enk was assayed usingfluorescamine. Emission spectra were measured on a SLM 48000 fluorimeterusing 380 nm excitation (λ=488 nm). Photorelease of Leu-Enk wasquantitated after 5 minutes of illumination.

Example 3 Solid Phase Synthesis of PCB-Polypeptides

[0246] PCB-amino acids were incorporated into polypeptides bysolid-support peptide synthesis. Standard method for employing baselabile fluorenylmethyloxy (Fmoc) group for the protection of α-aminofunction and acid labile t-butyl derivatives for protection ofα-carboxyl and reactive side chains were used. Synthesis was carried outon a polyamide-type resin. Amino acids were activated for coupling assymmetrical anhydrides or pentafluorophenyl esters (E. Atherton et al.,Solid Phase Peptide Synthesis, IRL Press, Oxford, 1989). The PCB-aminoacid for site-specific incorporation into polypeptide chain wasderivatized with PCB moiety. Side chain PCB-derivatives, likeε-amino-Lys, side chain PCB-AA esters of Glu and Asp, and esters of Ser,Thr and Tyr, were incorporated within the polypeptide. These PCB-aminoacids were stable during solid phase peptide synthesis, in 20%piperidine/DMF (Fmoc removal) and 1-95% trifluoroacetic acid (t-Bu,t-Boc removal, cleavage of the peptide from polyamide resin) (E.Atherton et al., Solid Phase Peptide Synthesis, IRL Press, Oxford,1989).

Example 4 Detection and Isolation of Nascent Proteins

[0247] Misaminoacylation of tRNA:

[0248] The general strategy used for generating misaminoacylated tRNA isshown in FIG. 10 and involves truncation of tRNA molecules, dinucleotidesynthesis, aminoacylation of the dinucleotide and ligase mediatedcoupling.

[0249] Truncated tRNA molecules were generated by periodate degradationin the presence of lysine and alkaline phosphatase basically asdescribed by Neu and Heppel (J. Biol. Chem. 239:2927-34, 1964). Briefly,4 mmoles of uncharged E. coli tRNA^(Lys) molecules (Sigma Chemical; St.Louis, Mo.) were truncated with two successive treatments of 50 mMsodium metaperiodate and 0.5 M lysine, pH 9.0, at 60° C. for 30 minutesin a total volume of 50 μl. Reaction conditions were always above 50° C.and utilized a 10-fold excess of metaperiodate. Excess periodate wasdestroyed treatment with 5 μl of 1M glycerol. The pH of the solution wasadjusted to 8.5 by adding 15 μl of Tris-HCl to a final concentration of0.1 M. The reaction volume was increased to 150 μl by adding 100 μl ofwater. Alkaline phosphatase (15 μl, 30 units) was added and the reactionmixture incubated again at 60° C. for two hours. Incubation was followedby ethanol precipitation of total tRNA, ethanol washing, drying thepellet and dissolving the pellet in 20 μl water. This process wasrepeated twice to obtain the truncated tRNA.

[0250] Dinucleotide synthesis was carried out basically as performed byHudson (J. Org. Chem. 53:617-24, 1988), and can be described as a threestep process, deoxycytidine protection, adenosine protection anddinucleotide synthesis.

[0251] Deoxycytidine Protection:

[0252] All reaction were conducted at room temperature unless otherwiseindicated. First, the 5′ and 3′ hydroxyl groups of deoxycytidine wereprotected by reacting with 4.1 equivalents of trimethylsilyl chloridefor 2 hours with constant stirring. Exocyclic amine function wasprotected by reacting it with 1.1 equivalents of Fmoc-Cl for 3 hours.Deprotection of the 5′ and 3′ hydroxyl was accomplished by the additionof 0.05 equivalents of KF and incubation for 30 minutes. The resultingproduct was produced at an 87% yield. Phosphate groups were added byincubating this compound with 1 equivalent of bis-(2-chlorophenyl)phosphorochloridate and incubating the mixture for 2 hours at 0° C. Theyield in this case was 25-30%.

[0253] Adenosine Protection:

[0254] Trimethylsilyl chloride (4.1 equivalents) was added to adenosineresidue and incubated for 2 hours, after which, 1.1 equivalents ofFmoc-Cl introduced and incubation continued for 3 hours. The TMS groupswere deprotected with 0.5 equivalents of fluoride ions as describedabove. The Fmoc protected adenosine was obtained in a 56% yield. Tofurther protect the 2′-hydroxyl, compound 22 was reacted with 1.1equivalents of tetraisopropyl disiloxyl chloride (TIPDSCl₂) for 3 hourswhich produces compound 23 at a 68-70% yield. The compound was convertedto compound 24 by incubation with 20 equivalents of dihydropyran and0.33 equivalents of p-toluenesulfonic acid in dioxane for about 4-5hours. This compound was directly converted without isolation by theaddition of 8 equivalents of tetrabutyl ammonium fluoride in a mixtureof tetrahydro-furan, pyridine and water.

[0255] Dinucleotide Synthesis:

[0256] The protected deoxycytidine, compound 20 (FIG. 19), and theprotected adenosine were coupled by the addition of 1.1 equivalents of2-chlorophenyl bis-(1-hydroxy benzotriazolyl) phosphate intetrahydrofuran with constant stirring for 30 minutes. This was followedby the addition of 13 equivalents of protected adenosine in the presenceof N-methylimidazole for 30 minutes. The coupling yield was about 70%and the proton NMR spectrum of the coupled product is as follows: (δ8.76 m, 2H), (δ 8.0 m, 3H), (δ 7.8 m, 3H) (δ 7.6 m, 4H), (δ 7.5 m, 3H),(δ 7.4 m, 18H), (δ 7.0 m, 2H), (δ 4.85 m, 14H), (δ 4.25 m, 1H); (δ 3.6m, 2H), (δ 3.2 m, 2H) (δ 2.9 m, 3H), (δ 2.6 m, 1H), (δ 2.0-1.2 m, 7H).

[0257] Aminoacylation of the dinucleotide was accomplished by linkingthe Nα-protected Nε-PCB-lys, to the dinucleotide with an ester linkage.First, the protected amino acid was activated with 6 equivalents ofbenzotriazol-1-yl-oxy tris-(dimethylamino) phosphonium hexafluorophosphate and 60 equivalents of 1-hydroxybenzotriazole intetrahydrofuran. The mixture was incubated for 20 minutes withcontinuous stirring. This was followed with the addition of 1 equivalentof dinucleotide in 3 equivalents N-methylimidazole, and the reactioncontinued at room temperature for 2 hours. Deprotection was carried outby the addition of tetramethyl guanidine and 4-nitrobenzaldoxime, andcontinuous stirring for another 3 hours. The reaction was completed bythe addition of acetic acid and incubation, again with continuousstirring for 30 minutes at 0° C. which produced the aminoacylateddinucleotide.

[0258] Ligation of the tRNA to the aminoacylated dinucleotide wasperformed basically as described by T. G. Heckler et al. (Tetrahedron40: 87-94, 1984). Briefly, truncated tRNA molecules (8.6 O.D.₂₆₀units/ml) and aminoacylated dinucleotides (4.6 O.D.₂₆₀ units/ml), wereincubated with 340 units/ml T4 RNA ligase for 16 hours at 4° C. Thereaction buffer included 55 mM Na-Hepes, pH 7.5, 15 MM MgCl₂, 250 μMATP, 20 μg/ml BSA and 10% DMSO. After incubation, the reaction mixturewas diluted to a final concentration of 50 mM NaOAc, pH 45, containing10 mM MgCl₂. The resulting mixture was applied to a DEAE-cellulosecolumn (1 ml), equilibrated with 50 mM NaOAc, pH 45, 10 mM MgCl₂, at 4°C. The column was washed with 0.25 mM NaCl to remove RNA ligase andother non-tRNA components. The tRNA-containing factions were pooled andloaded onto a BD-cellulose column at 4° C., that had been equilibratedwith 50 mM NaOAc, pH 4.5, 10 mM MgCl₂, and 1.0 M NaCl. Unreacted tRNAwas removed by washes with 10 ml of the same buffer. Puremisaminoacylated tRNA was obtained by eluting the column with buffercontaining 25% ethanol.

[0259] Preparation of extract: Wheat germ embryo extract was prepared byfloatation of wheat germs to enrich for embryos using a mixture ofcyclohexane and carbon tetrachloride (1:6), followed by drying overnight(about 14 hours). Floated wheat germ embryos (5 g) were ground in amortar with 5 grams of powdered glass to obtain a fine powder.Extraction medium (Buffer I: 10 mM tris-acetate buffer, pH 7.6, 1 nMmagnesium acetate, 90 mM potassium acetate, and 1 mM DTT) was added tosmall portions until a smooth paste was obtained. The homogenatecontaining disrupted embryos and 25 ml of extraction medium wascentrifuged twice at 23,000×g. The extract was applied to a sephadexG-25 fine column and eluted in Buffer II (10 mM tris-acetate buffer, pH7.6, 3 mM magnesium acetate, 50 mM potassium acetate, and 1 mM DTT). Abright yellow band migrating in void volume and was collected (S-23) asone ml fractions which were frozen in liquid nitrogen.

[0260] Preparation of Template:

[0261] Template DNA was prepared by linearizing plasmid pSP72-bop withEcoRI. Restricted linear template DNA was purified by phenol extractionand DNA precipitation. Large scale mRNA synthesis was carried out by invitro transcription using the SP6-ribomax system (Promega; Madison,Wis.). Purified mRNA was denatured at 67° C. for 10 minutes immediatelyprior to use.

[0262] Cell-Free Translation Reactions: The incorporation mixture (100μl) contained 50 μl of S-23 extract, 5 mM magnesium acetate, 5 mMtris-acetate, pH 7.6, 20 mM Hepes-KOH buffer, pH 7.5; 100 mM potassiumacetate, 0.5 mM DTT, 0.375 mM GTP, 2.5 mM ATP, 10 mM creatine phosphate,60 μg/ml creatine kinase, and 100 μg/ml mRNA containing the geneticsequence which codes for bacteriorhodopsin. Misaminoacylated PCB-lysinewas added at 170 μg/ml and concentrations of magnesium ions and ATP wereoptimized. The mixture was incubated at 25° C. for one hour.

[0263] Isolation of Nascent Proteins Containing PCB-Lysine:

[0264] Streptavidin coated magnetic Dynabeads M-280 (Dynal; Oslo,Norway), having a binding capacity of 10 μg of biotinylated protein permg of bead. Beads at concentrations of 2 mg/mL were washed at least 3times to remove stabilizing BSA. The translation mixture containingPCB-lysine incorporated into nascent protein was mixed with streptavidincoated beads and incubated at room temperature for 30 minutes. Amagnetic field was applied using a magnetic particle concentrator (MPC)(Dynal; Oslo, Norway) for 0.5-1.0 minute and the supernatant removedwith pipettes. The reaction mixture was washed 3 times and the magneticbeads suspended in 50 μof water.

[0265] Photolysis was carried out in a quartz cuvette using a Black-Raylong wave UV lamp, Model B-100 (UV Products, Inc.; San Gabriel, Calif.).The emission peak intensity was approximately 1100 μW/cm² at 365 nm.Magnetic capture was repeated to remove the beads. Nascent proteinsobtained were quantitated and yields estimated at 70-95%.

Example 5 In Vitro Synthesis of Nascent Proteins using PhotocleavableConjugates

[0266] Post-Aminoacylation Linkage:

[0267] A schematic representation of the steps involved in incorporationof PCB-amino acid for the detection and/or isolation of targets usingpost-aminoacylation linkage is shown in FIG. 10. E. coli tRNA^(Lys)(Sigma Chem.; St. Louis, Mo.) was aminoacylated with lysine (A. E.Johnson et al., Proc. Natl. Acad. Sci. USA 75:3075, 1978). The NHS esterof PCB (compound 13) dissolved in dimethyl sulphoxide, was added at 0°C. to the solution of Lys-tRNA^(Lys) and the modified tRNA purifiedusing benzoylated DEAE-cellulose column (U. C. Kreig et al., Proc. Natl.Acad. Sci. USA 83:8604, 1986). mRNA was translated in a cell-free,wheat-germ system as described by Sonar et al. (Biochem. 32:13777,1993). Nascent proteins containing PCB-lysine were purified by acetoneprecipitation to remove PCB-lysyl tRNA followed by magnetic capture ofnascent proteins containing PCB-lysine using streptavidin coatedmagnetic beads. Material obtained after magnetic capture was irradiatedfor 10 minutes to release nascent protein.

Example 6 Synthesis of Photocleavable Conjugates—PCB Nucleotides

[0268] Synthesis of PCB-dUTP (FIG. 13A):

[0269] 5-(3-Aminoallyl)-dUTP ammonium salt (“31”) (Sigma Chemical; St.Louis, Mo.) (10 mg or 16.6 μmol) was dissolved in 200 μl of 0.1N NaHCO₃.To this solution was added a solution of PCB-NHS (compound 13; 12.5 mgor 1 eq.) in 100 μl of DMF. The reaction mixture was stirred overnightat room temperature, concentrated, and purified by reverse-phasesemi-preparative HPLC (Novapak C₁₈ column; Waters Chromatography;Marlboro, Mass.) using a 10-50% linear gradient of acetonitrile (B) in 5mM triethylammonium acetate (A) over 30 minutes. Fractions containingPCB-dUTP were pooled, lyophilized, and redissolved in TE buffer (pH 7.4)to a concentration of 5 mM, and the solution used for enzymaticincorporation into nucleic acids (yield about 56%). Similar procedureswere used to prepare PCB-UTP, PCB-(d)ATP, and PCB-(d)CTP, using5-(3-aminohexyl)-(deoxy)cytidine triphosphate, respectively.

Example 7 Synthesis of Photocleavable Conjugates—PCB Phosphoramidites

[0270] Synthesis of PCB-phosphoramidite (FIG. 13B):

[0271] 5-(-5-biotinamidocapro-amidomethyl)-2-nitroacetophenone (“37”)(534 mg or 1 mmol) was made anhydrous by coevaporation with pyridine(3×2 ml) and dissolved in 5 ml of pyridine. 4,4′-dimethoxytritylchloride (406 mg or 1.2 eq) and 4-dimethylaminopyridine (6 mg or 0.05eq) were added and the resulting solution stirred at room temperaturefor 24 hours. Ten ml of CHCl₃ and 20 ml of 0.1N aqueous NaHCO₃ wereadded, the layers formed separated and the organic layer dried,evaporated to dryness and purified on a silica gel column using 0-5%step gradient of MeOH in CHCl₃ to give 576 mg (about 69%) of compound38. Intermediate 38 (836 mg or 1 mmol) was dissolved in 8 ml of 95%EtOH. The solution was cooled to 0° C. and vigorously stirred. To thesolution was added NaBH₄ (19 mg or 2 eq.) in portions and the solutionstirred for an additional 2 hours at room temperature. The reaction wasquenched with 2 ml of acetone, 10 ml of CHCl₃ and 10 ml of 0.1N aqueousNaHCO₃ were added, the layers were separated, the organic layer wasdried and evaporated to dryness to give 704 mg (about 84%) of compound39 which was used without additional purification. Compound 39 (838 mgor 1 mmol) was dissolved in a mixture of CHCl₃ (5 ml) anddiisophrophylethylamine (0.68 ml or 4 eq.). To this solution was added2-cyanoethyl-N-,N-diisophropylchlora-phosphoramidite (225 μl or 1 eq.)and the solution was stirred at room temperature for 1 hour. Ethylacetate (5 ml) was added and the solution was washed with an NaClsolution (3×1 ml) and H₂O (2 ml). Dried solvents were removed in vacuoand purified on a silica gel column using step gradient of triethylaminein CH₂Cl₂ with a yield of 789 mg (about 74%).

Example 8 Chemical Synthesis of Oligonucleotides usingPCB-Phosphoramidites

[0272] Automated Synthesis and Purification of Oligonucleotides Using 5′PCB-phosphoramidite:

[0273] A 0.1M solution of 5′-PCB-phosphoramidite in anhydrousacetonitrile was prepared. The bottle with the solution was placed inthe additional phosphoramidite port of the Applied Biosystem 392 DNA/RNAsynthesizer. An oligodeoxynucleotide sequence was programmed andsynthesized using 40 nmol CE column and standard synthesis protocol. Theonly modification necessary was extended detritylation (180 s) necessaryfor removal of trityl group from N¹ position of biotin. After synthesis,5′-PCB-oligodeoxynucleotide was cleaved from solid support anddeprotected by treatment with concentrated ammonia for 16 hours at 50°C. The crude oligonucleotide was freeze-dried and dissolved in 1 ml ofphosphate buffer, pH 7.4. To this solution was added a suspension ofmonomeric avidin agarose beads (Sigma Chemical; St. Louis, Mo. ) (40nmoles), and the mixture was incubated at room temperature for 1 hour.The suspension was filtered and washed with 3×1 ml phosphate buffer,resuspended in 3 ml of phosphate buffer and illuminated as describedwith gentle stirring for 10 minutes. The mixture was filtered, filtratefreeze-dried and redissolved (yield=3.8 OD₂₆₀).

Example 9 Enzymatic Synthesis of DNA and RNA using PCB-Nucleotides

[0274] Several enzymatic and chemical methods are available forbiotinylation of nucleic acid probes. Enzymatic methods forincorporation of PCB-nucleotides into DNA include nick translation andreplacement synthesis using T4 DNA polymerase. Terminal labeling of DNAcan also be performed using terminal deoxynucleotidyl transferase. ForPCB-labeling of RNA several RNA polymerase enzymes can be used. Nicktranslation was performed in the presence of PCB-dCTP based on themethods developed for biotinylation (P. R. Langer et al., Proc. Natl.Acad. Sci. USA 78:6633, 1981). Enzymatic tailing was used for double-and single-stranded DNA molecules. PCB nucleotides were added onto the3′-end of the DNA. Biotinylated probes with internal biotin moietiesform less stable hybrids than probes with external biotins and that thebiotinylated probes synthesized in this manner have greater sensitivitythan probes that are singly biotinylated at 5′-end (E. P. Diamandis etal., Clin. Chem. 37:625, 1991).

[0275] Preparation of PCB-labeled RNA:

[0276] PCB-labeling of RNA was achieved in a standard phage T7 RNApolymerase transcription system using the PCB-UTP. To preparesingle-stranded, biotinylated RNA as a probe, the appropriate DNAsequence was cloned into an appropriate vector which contains the T7promoter upstream from the polylinker region. After linearization of theDNA clone downstream from the cloned insert, the RNA transcript ofdefined length was produced by the T7 RNA polymerase using ATP, CTP, GTPand PCB-UTP as substrates.

Example 10 Isolation of Hematopoietic Cells for Autologous Bone MarrowTransplantation.

[0277] Bone marrow is collected from the posterior iliac crest of normalhealthy and leukemic patients into heparin. Low-density mononuclearcells are separated by sedimentation on Ficoll-Hypaque (Sigma Chemical;St. Louis, Mo.). CD34⁺ cells are isolated using PCB-labeled anti-CD34monoclonal antibodies (My10). Mononuclear marrow cells are placed atconcentrations of 10⁶/ml in Iscove's Modified Dulbeco's Medium (IMDM;Irvine Scientific; Santa Anna, Calif.) with 20% FCS. Cells are culturedovernight under tissue culture conditions to remove adherent cells.Nonadherent cells are collected, washed twice in cold phosphate bufferedsaline (PBS), and diluted in PBS to 10⁷/ml. PCB-labeled anti-CD34antibodies are added to the cell suspension at 5 μ/ml and incubated at4° C. for one hour with gentle intermittent mixing. After incubation,cells are washed twice in 5%-FCS/PBS and resuspended in the same volume.Streptavidin coated magnetic beads (Dynabeads; Oslo, Norway) are addedto the suspension which is incubated at 4° C. for one hour with mixing.Beads and their associated cells are subjected to a magnet and separatedfrom the suspension and placed in 5%-FCS/PBS. Photocleavage is carriedout by irradiating the beads for 4 minutes with a long-wavelength,UV-lamp (Black Ray XX-15 UV lamp; UVP Inc; San Gabriel, Calif.) at adistance of 15 cm (emission peak 365 nm, lamp intensity=1.1 mW/cm² at adistance of 31 cm). Released beads are isolated by magnetic capture. Thecell suspension is assayed for CD34⁺ cells by staining withFTC-conjugated My10 antibody followed with FACS analysis and determinedto be greater than 95% CD34 cells.

Example 11 Determination of the In Vivo Half-life of a PharmaceuticalComposition

[0278] Cell-free translation reactions are performed by mixing 10 μl ofPCB-coumarin amino acid-tRNA^(leu), prepared by chemicalmisaminoacylation as described above and suspended in TE at 1.7 mg/ml),50 μl of S-23 extract, 10 μl water and 10 μl of a solution of 50 mMmagnesium acetate, 50 mM Tris-acetate, pH 7.6, 200 mM Hepes-KOH buffer,pH 7.5; 1 M potassium acetate, 5 mM DTT, 3.75 mM GTP, 25 mM ATP, 100 mMcreatine phosphate and 600 μg/ml creatine kinase. This mixture is kepton ice until the addition of 20 μl of 500 μg/ml human IL-2 mRNA(containing 26 leucine codons) transcribed and isolated from recombinantIL-2 cDNA. The mixture is incubated at 25° C. for one hour and placed onice. One hundred μl of streptavidin coated magnetic Dynabeads (2 mg/ml)are added to the mixture which is placed at room temperature for 30minutes. After incubation, the mixture is centrifuged for 5 minutes in amicrofuge at 3,000×g or, a magnetic field is applied to the solutionusing a MPC. Supernatant is removed and the procedure repeated threetimes with TE. The final washed pellet is resuspended in 50 μl of 50 mMTris-HCl, pH 75 and transferred to a quartz cuvette. UV light from aBlack-Ray long wave UV lamp is applied to the suspension forapproximately one second. A magnetic field is applied to the solutionwith a MPC for 1.0 minute and the supernatant removed with a pipette.The supernatant is sterile filtered and mixed with equal volumes ofsterile buffer containing 50% glycerol, 1.8% NaCl and 25 mM sodiumbicarbonate. Protein concentration is determined by measuring theO.D.₂₆₀.

[0279] 0.25 ml of the resulting composition is injected iv. into thetail vein of 2 Balb/c mice at concentrations of 1 mg/ml. Two controlmice are also injected with a comparable volume of buffer. At varioustime points (0, 5 minutes, 15 minutes, 30 minutes, 60 minutes, 2 hoursand 6 hours), 100 μl serum samples are obtained from foot pads and addedto 400 μl of 0.9% saline. Serum sample are added to a solution ofdynabeads (2 mg/ml) coated with anti-coumarin antibody and incubated atroom temperature for 30 minutes. A magnetic field is applied to thesolution with a MPC for 1 minute and the supernatant removed with apipette. Fluorescence at 470 nm is measured and the samples treated withmonoclonal antibody specific for rat IL-2 protein. IL-2 protein contentis quantitated for each sample and equated with the amount offluorescence detected. From the results obtained, in vivo IL-2 half-lifeis accurately determined.

Example 12 Polymerase Chain Reactions with PCB

[0280] The steps involved in the PCR amplification DNA sequences usingPCB are shown in FIG. 12. The experimental method described below isbased on a combination of protocols described (Y. Lo et al., Nucl. AcidsRes. 16:719, 1988; R. K. Saiki et al., Sci. 239:487, 1988). PCB-dCTPsynthesized was added (50 μM) to the mixture of dATP, dGTP, dTTP (all200 μM) and dCTP (150 μM). The reaction mixture consisted of target DNAsource (total genomic DNA isolated), flanking primers and thethermostable polymerase (Taq polymerase). The reaction mixture wassubjected to 25-30 cycles of amplification. Samples were heated from70-95° C. for a 1 minute period to denature DNA and cooled to 40° C. for2 minutes to anneal the primers. Samples were again heated to 70° C. for1 minute to activate the polymerase and incubated at this temperaturefor 0.5 minutes to extend the annealed primers. After the last cycle,samples were incubated for an additional 5 to 10 minutes at 37° C. toensure that the final extension was complete. Magnetic capture of thenucleic acids was performed using streptavidin coated magnetic beads.The captured material was washed with appropriate buffers andresuspended at the desired concentration. Samples were illuminated for10 minutes to release the PCR product in an unmodified form.

[0281] Nucleic acids in either immobilized form or in solution form weredetected or separated (purified) using PCB-labeled nucleic acid probes.Hybridization was carried out to obtain DNA:DNA, DNA:RNA and RNA:RNAhybrids. These experiments involve first the hybridization withPCB-labeled probes followed by capturing the hybrids using streptavidincoated immobilized supports. These hybrids were washed free of initialundesired components and were released from the immobilized supportusing irradiation (G. Gebeyehu et al., Nucl. Acids Res. 15:4513, 1987;T. Ito et al., Nucl. Acids Res. 20:3524, 1992). Hybridization of ssDNAmolecules with PCB-probe involved incubation of these components in ahybridization buffer at 42° C. for 30 minutes. Hybridization conditionswere optimized for each probe and experimental system. PCB-labeledprobes had lower melting temperatures than radiolabeled probes andrequire slightly modified hybridization conditions. These hybrids wereselectively removed from the reaction components using immobilizedstreptavidin (Dynabeads M280 streptavidin). Photochemical release ofcomplexes resulted in the isolation of pure hybrid.

Example 13 Synthesis of PCB—Liposomes

[0282] Incorporation of PCB-Lipids into Liposomes:

[0283] PCB-lipids were mixed with conventional lipids inchloroform:methanol at a ratio of 2:1. The lipid mixture was evaporatedto dryness under nitrogen and the dried lipids suspended in DMSO assolvent to a final concentration of 1 mg/ml. Liposomes were sonicationunder nitrogen in an ice-cold chamber for 10 minutes. The resultingsuspension was centrifuged for 20 minutes at 10,000 rpm and thesupernatant containing PCB-liposomes was ready for use. Satisfactoryresults were obtained with as little as 5% (mol equivalent) PCB-lipids.The structural chemical formulas for PCB-phosphatidylathanolamine andPCB-phosphatidylserine are shown in FIG. 16.

Example 14 PCB for In Situ Hybridization

[0284] The general methodology for in situ hybridization reactions canbe divided into sample preparation, selection of indicator molecule andprobe, hybridization, washing, and autoradiography and detection.

[0285] Sample Preparation:

[0286] Frozen tissue sections of 5 to 6 μm are mounted on gelatin coatedmicroscope slides and air dried for 30 min prior to fixation. This isfollowed by fixation of DNA or RNA using either glutaraldehyde orparaformaldehyde. During these fixing steps optional denaturing steps(e.g. 100° C. for 5 minutes) followed by quick immersion in ice coldbuffer (necessary if dsDNA or dsRNA is the target of the reaction) canbe introduced. In case of cells and cell-cultures, these cells (1×10⁶cells) are deposited on gelatin coated slides by cytocentrifugation orsmearing. The cells are air dried and fixed for DNA or RNA.

[0287] Selection of Indicator Molecule and Probe:

[0288] Although isotopic detection offers several advantages over theuse of non-isotopic methods, the latter can be used effectively. Labeledprobes can be generated by a variety of techniques ranging fromsynthetic oligonucleotides to excised plasmid inserts.

[0289] Hybridization:

[0290] ISH follows the same general principles as a solution and filterhybridization. Standard reaction temperatures are approximatelyT_(m)−25° C. (T_(m) is the temperature at which 50% of hybridsdissociate). The reaction temperature is reduced to a level compatiblewith the preservation of histological detail by the addition of 50%formamide to the hybridization mixture. Thus, for typical DNA-DNAhybridization reactions, the temperature is 37° C., for RNA-DNA 44° C.,and for RNA-RNA 50°. The surface of the microscope slide supporting thesample is gently blown by a stream of air, and the final hybridizationmix is pipetted over the surface. The film is then incubated flat in abath of paraffin oil for the required time and temperature.

[0291] Washing:

[0292] The paraffin oil is drained and excess of oil is removed bywashing twice with chloroform, and the slides are air dried. A highstringency wash is given to reduce background.

[0293] Autoradiography and Detection:

[0294] Detection is carried out either using X-ray film or emulsioncoated cover-slips in cases of radioactive isotopically labeled probes,and other methods in the case of enzymatic detection methods.

Example 15 Isolation of Different Populations Cells with Agents whichPhotocleaved at Distinct Wavelengths.

[0295] Two distinct conjugates are created, each with a differentantigen-specific antibody coupled to a different bioreactive agent.Conjugate A comprises compound 30 (FIG. 21), a PCB bioreactive agent,coupled to an antibody specific for the cell surface marker CD34 (a stemcell marker), and will photocleave with radiation at 300 nm. Conjugate Bcomprises compound 25 (FIG. 19), a PCB bioreactive agent, coupled to anantibody specific for the cell surface marker CD3 (a T cell marker), andwill photocleave with radiation at 400 nm.

[0296] Conjugates A and B are incubated, in duplicate, with samples ofperipheral blood obtained from healthy human volunteers. Incubations areperformed at room temperature (22° C.) with gentle rocking to providemaximal antibody-antigen contact. After a 30 minute incubation, cellsare placed in 100 mm tissue culture dishes coated with streptavidin andincubated for an additional 30 minutes. Upon streptavidin-biotinbinding, plates are gently washed in PBS to remove any cells which donot adhere.

[0297] After washing, one set of plates is treated with electromagneticradiation at 300 nm and the released cells collected. This set is thentreated with electromagnetic radiation at 400 nm and the cells releasedat this frequency collected. A second set of plates is treated withradiation at 400 nm, released cells are collected, the plates are againtreated at 300 nm and the released cells again collected. By determiningthe number of cells collected after each treatment and from each set ofplates, the number of cells in a sample of peripheral blood which carrythe cell surface marker for CD34, CD3, and both CD34 and CD3 isdetermined.

[0298] Other embodiments and uses of the invention will be apparent tothose skilled in the art from consideration of the specification andpractice of the invention disclosed herein. It is intended that thespecification and examples be considered exemplary only, with the truescope and spirit of the invention being indicated by the followingclaims.

1. A method, comprising: a) providing a tRNA molecule, nucleic acidderived from a biological sample, and a marker; b) aminoacylating saidtRNA molecule with said marker to create a misaminoacylated tRNA; c)introducing said misaminoacylated tRNA and said nucleic acid derivedfrom a biological sample into a translation system under conditions suchthat said marker is incorporated into a nascent protein; and d)screening for disease, said screening comprising detecting said marker.2. The method of claim 1, wherein the nascent protein is selected fromrecombinant gene products, gene fusion products, enzymes, cytokines,carbohydrate and lipid binding proteins, nucleic acid binding proteins,hormones, immunogenic proteins, human proteins, viral proteins,bacterial proteins, parasitic proteins and fragments and combinationsthereof
 3. The method of claim 1, wherein the translation systemcomprises a cellular or cell free translation system.
 4. The method ofclaim 3, wherein the cellular translation system is selected from thegroup consisting of tissue culture cells, primary cells, cells in vivo,isolated immortalized cells, human cells and combinations thereof. 5.The method of claim 3, wherein the cell-free translation system isselected from the group consisting of Escherichia coli lysates, wheatgerm extracts, insect cell lysates, rabbit reticulocyte lysates, frogoocyte lysates, dog pancreatic lysates, human cell lysates, mixtures ofpurified or semi-purified translation factors and combinations thereof.6. The method of claim 1, wherein the tRNA molecule is aminoacylated bychemical or enzymatic misaminoacylation.
 7. The method of claim 1,wherein said disease is selected from the group consisting ofinfections, neoplasias and genetic disorders.
 8. The method of claim 1,wherein said biological sample is selected from the group consisting ofblood, serum, tissue, urine, stool, nasal cells and spinal fluid.
 9. Themethod of claim 1, wherein prior to step (c) said nucleic acid isamplied in a polymerase chain reaction.
 10. The method of claim 1,wherein said marker comprises a detectable label.
 11. The method ofclaim 1, wherein said marker comprises a coupling agent.
 12. The methodof claim 11, wherein said coupling agent is selected from the groupconsisting of biotin and derivatives thereof.
 13. The method of claim11, wherein said coupling agent comprises an antigenic site.
 14. Themethod of claim 11, wherein said coupling agent binds to surfaces, saidsurfaces selected from the group consisting of resin surfaces, beadsurfaces, ceramic surfaces, glass surfaces, particle surfaces, andpolymer surfaces.
 15. The method of claim 1, wherein said marker is aphotocleavable marker.
 16. The method of claim 15, wherein saidphotocleavable marker is photocleavable biotin.
 17. The method of claim16, wherein said photocleavable marker comprises an amino acid coupledvia a photocleavable linkage to a detectable label.
 18. The method ofclaim 15, wherein said photocleavable marker comprises an amino acidcoupled via a photocleavable linkage to a coupling agent.
 19. The methodof claim 1, wherein two or more different misaminoacylated tRNAs areintroduced to the translation system at step (c).
 20. The method ofclaim 1, wherein said detecting of step (d) comprises using anelectromagnetic field.